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2007 Regulation of FOXO stability and activity by E3 ligase Wei Fu University of South Florida

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Regulation of FOXO Stability and Activity by MDM2 E3 Ligase

By

WEI FU

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology and Biology College of Medicine University of South Florida

Major Professor: Wenlong Bai, Ph.D. George Blanck, Ph.D. Jiandong Chen, Ph.D. Santo V. Nicosia, M.D John C.M. Tsibris, Ph.D.

Date of Approval: Nov., 2007

Key words: Forkhead Transcriptional Factors, MDM2, Ubiquitination, Degradation,

©Copyright 2007, Wei Fu

Dedication

To my parents, my in-laws, my brother, my husband, Zhigang and my little pumpkin, caroline.

Without their love, understanding and support, I could

not have done even one piece of it.

Acknowledgement

I would like to extend my sincere appreciation to Dr. Wenlong Bai for guiding me in the development of this project and the invaluable support from him at each step throughout my graduate studies.

My appreciation also goes to Dr. Santo V. Nicosia for being helped in every way.

I would like to thank my adviser and committee members, Drs Bai, Blanck, Chen,

Nicosia and Tsibris for their guidance, valuable suggestion and their word of encouragement.

My appreciation also goes to Dr. Yanping Zhang for willing to be my outside chair.

Thanks to Dr. Jiandong Chen and the members in his lab, in particular to Lihong Chen, for their generous supply of the cell lines, plasmids, antibodies and priceless technical assistant.

Thanks to Dr. Xiaohong (Mary) Zhang for all her kindly help.

Thanks to all the colleagues in Dr. Bai’s and Dr. Zhang’s Lab: Qiuping Ma, Yonghua

Yang, Pengfei Li, Heehyoung Lee, Feng Jiang, Chunrong Li, Betty Bean, Xiaohui Zhang,

Junying Bao, Lucy Hou, Zheng Shen, Guoqing Liu, Latasha Lee, Ushiwa Jinwal, Tung

Hsieh, Yingtao Zhang, Sarah Yong, Huiqin Dong and Mu Zhang for sharing their opinions and joy with me.

Thanks to Dr. Patricia Kruk and all the members in her lab.

Thanks to Dr. Jin Cheng and the members in his lab, especially to Mei Sun, Zengqiang

Yuan and Hua Yang for valuable suggestion.

I would like thank to all the members in the Department of Pathology and Cell Biology.

Addtionally, I would like to thank everyone in Graduate Affairs Office, especially to Dr.

Barber, Dr. Joseph Krzanowski, Ms Kathy Zahn, Ms Susan Chapman.

Thanks to Moffitt & College of Medicine core facility for some analysis.

Thanks to all my friends who guide me, encourage me and help me on the way.

This thesis is dedicated to my family. Word cannot express my grateful feeling. I want to say thanks to my husband, Zhigang, for his loving and my daughter, Caroline, for her shining smile that let me feel I am the happiest mother in the world. I would like to thank my parents and my brother, for their life-long loving. I would also like to thank my in-laws for their kindly help.

The research in this dissertation has been partially supported by Predoctoral Fellowship from American Heart Association.

Thanks to Dr. Tsibris and Dr. Aslam for reading this thesis and their valuable suggestions.

Table of Contents

List of Figures...... vi

List of Tables...... x

Abstract ...... xi

INTRODUCTION...... 1

1. FOXO Family...... 1

1.1. FOX Family...... 1

1.2. FOXO Family...... 4

1.2.1. Classification of FOXO Factors...... 5

1.2.2. Domain Structure of FOXO...... 7

1.3. Function of FOXO Factors...... 9

1.3.1. FOXO and Checkpoint and DNA Repair ...... 10

1.3.1.1. FOXO and G1/S Checkpoint...... 10

1.3.1.2. FOXO and G2/M Checkpoint and DNA Repair...... 11

1.3.2. FOXO and Apoptosis...... 12

1.3.3. FOXO and Atrophy...... 13

1.3.4. FOXO and ROS Detoxification in Stem Cells ...... 14

1.3.5. FOXO and Tissue Differentiation...... 15

1.3.6. FOXO and Glucose and Energy Metabolism...... 16

i 1.3.7. FOXO and Longevity...... 17

1.4. Posttranslational Modifications of FOXO ...... 18

1.4.1. of FOXO...... 18

1.4.1.1. PKB and FOXO...... 18

1.4.1.2. SGK and FOXO ...... 20

1.4.1.3. IκB Kinase and FOXO3A ...... 21

1.4.1.4. CK1 and FOXO...... 21

1.4.1.5. CDK2 and FOXO ...... 21

1.4.1.6. JNK and FOXO...... 21

1.4.1.7. MST and FOXO3A...... 22

1.4.2. and Deacetylation of FOXO ...... 23

1.4.2.1. Acetylation and Deacetylation...... 23

1.4.2.2. Acetylation of FOXO ...... 23

1.4.2.2.1. P300 and CBP...... 23

1.4.2.2.2. P300/CBP and FOXO Acetylation...... 25

1.4.2.3. Deacetylation of FOXO by HDAC...... 26

1.4.2.3.1. SIRT1 ...... 26

1.4.2.3.2. SIRT1 and FOXO Deacetylation...... 28

1.4.3. Ubiquitination and Deubiquitination of FOXO ...... 30

1.4.3.1. Ubiquitination and Degradation of FOXO...... 30

1.4.3.2. Deubiquitination of FOXO...... 31

2. , and MDM2 as an E3 Ligase...... 32

2.1. Ubiquitin-proteasome System...... 32

2.1.1. Ubiquitin and Ubiqutination...... 32

2.1.2. Deubiquitination ...... 33

ii 2.1.3. Ubiquitination Machinery and Proteasome Pathway ...... 35

2.2. MDM2 as an Ubiquitin E3 Ligase ...... 37

2.2.1. General Information about MDM2...... 37

2.2.2. Functions of MDM2...... 38

2.2.2.1. MDM2 and Cell Cycle...... 38

2.2.2.2. MDM2 and Differentiation...... 38

2.2.2.3. MDM2 and Ribosome Biogenesis...... 39

2.2.2.4. MDM2 and ...... 40

2.2.2.5. MDM2 and Protein Ubiquitination and Degradation...... 40

2.2.3. Regulation of MDM2 E3 Activity...... 41

2.2.3.1. Sumoylation of MDM2...... 41

2.2.3.1.1. SUMO and Sumoylation...... 41

2.2.3.1.2. Sumoylation and MDM2...... 43

2.2.3.2. Ubiquitination and Degradation of MDM2...... 43

2.2.3.3. Phosphorylation of MDM2...... 44

2.2.3.3.1. DNA-PK, ATM and MDM2...... 44

2.2.3.3.2. PKB and MDM2...... 45

2.2.3.3.3. c-Abl and MDM2...... 46

2.2.3.3.4. CK2 and MDM2...... 46

3. Functional interaction among FOXO, , and MDM2...... 49

3.1. Interaction between MDM2 and p53...... 49

3.2. Interaction between p53 and FOXO ...... 50

HYPOTHESIS & OBJECTIVES ...... 53

MATERIALS AND METHODS ...... 54

1. Chemicals, Antibodies and Cell Lines ...... 54

iii 2. Plasmids...... 54

3. Transfections and Immunological Assays ...... 55

4. Ni-NTA Pull-down Assay ...... 57

5. In vitro Transcription Coupled Translations and GST (Glutathione

S-transferase) Pull-down Assays ...... 58

6. In Vitro Ubiquitination Assay...... 58

7. Apoptotic Analysis and Flow Cytometry ...... 59

RESULTS...... 60

1. MDM2 promotes the degradation of FOXO...... 60

2. MDM2 interacts with FOXO in vivo and in vitro ...... 69

3. The fork head box of FOXO and the region of MDM2 controlling

nuclear-cytoplasmic shuttling mediate the interaction between

MDM2 and FOXO...... 77

4. MDM2 promotes the ubiquitination of FOXO1 and FOXO3A ...... 88

5. MDM2 suppresses the expression of FOXO target and

protects cells from FOXO1-induced cell death...... 96

6. MDM2 transiently increases FOXO transcriptional activity...... 103

7. p53 induces transient increase in the transcriptional activity of

FOXO factors, which is followed by FOXO degradation in an

MDM2-dependent manner...... 108

8. Site-specific sumoylation of SIRT1 regulates FOXO1

transcriptional activity and stability ...... 116

9. Genistein-induced FOXO1 expression is blocked by MDM2

expression in H1299 cells...... 121

10. ARF promotes the MDM2-induced FOXO ubiquitination...... 123

iv DISCUSSION...... 125

1. MDM2 is the E3 of FOXO ...... 125

2. MDM2-promoted ubiquitination of FOXO depends on

phosphorylation on PKB sites...... 126

3. MDM2-promoted ubiquitination of FOXO happenes mainly in the

cytoplasm ...... 127

4. MDM2 affects both the transcriptional activity and ubiquitination of

FOXO ...... 128

5. Mammalian FOXO factors interact with p53 ...... 129

6. MDM2 regulates FOXO factors in a p53 independent way ...... 130

7. Different domains of MDM2 play different roles in the regulation of

p53 and FOXO factors (see Table 2) ...... 132

8. The sumoylation status of SIRT1 affects the stability and activity of

FOXO ...... 134

9. Genistein increases FOXO expression levels through down-

regulation of MDM2 ...... 134

10. ARF differentially affects E3 function of MDM2 toward different

substrates...... 135

SUMMARY AND PERSPECTIVES...... 136

REFERENCES...... 139

About the author...... End Page

v

List of Figures

Figure 1 Structure of human FOXO...... 8

Figure 2 The regulation of FOXO ...... 29

Figure 3 Ubiquitin-proteasome degradation pathway ...... 36

Figure 4 Structure of MDM2 and protein ...... 47

Figure 5 Stress-induced FOXO and p53 pathway ...... 52

Figure 6 Inverse correlation of MDM2 and FOXO3A protein expression in

different cancer cell lines ...... 62

Figure 7 Inverse correlation of MDM2 and FOXO protein expression in MEFs...... 63

Figure 8 Stably expressed MDM2 decreases the level of endogenous

FOXO3A expression...... 64

Figure 9 Knockdown of endogenous MDM2 by siRNA causes an increase in

endogenous FOXO3A protein ...... 65

Figure 10 Knockdown of endogenous MDM2 by shRNA increases the level of

ectopic FOXO1 protein ...... 66

Figure 11 MDM2 overexpression causes a decrease in Flag-FOXO1 protein,

which is blocked by MG132...... 67

Figure 12 MDM2 overexpression results in decrease in the half-life of the

FOXO1 protein ...... 68

Figure 13 Exogenous MDM2 and Flag-FOXO1 are colocalized in the nucleus

of MEF cells...... 70

vi Figure 14 Endogenous MDM2 and FOXO3A are colocalized in the nucleus of

H1299 cells...... 71

Figure 15 Interaction of ectopic FOXO1 and MDM2 in H1299 cells ...... 72

Figure 16 Interaction of ectopic FOXO3A and MDM2 in HEK293T cells...... 73

Figure 17 Interaction of endogenous MDM2 and FOXO1 in H1299/V138 cells ...... 74

Figure 18 Interaction of endogenous MDM2 and FOXO3A in HEK293T cells ...... 75

Figure 19 The interaction between FOXO1 and GST-MDM2 in vitro ...... 76

Figure 20 Diagram of different FOXO mutants used in this study ...... 79

Figure 21 Mapping of the MDM2-interacting domain of FOXO1 by

immunoprecipitation ...... 80

Figure 22 Mapping of the MDM2-interacting domain of FOXO3A by GST pull-

down assay...... 81

Figure 23 Alignment of FOXO members’ fork head box...... 82

Figure 24 Diagram of different MDM2 mutants used in this study...... 83

Figure 25 Mapping of the FOXO1-interacting domain of MDM2 by

coimmunoprecipitations in the absence of MG132...... 84

Figure 26 An N-terminal truncation mutant and an NLS-mutant of MDM2

interact with FOXO1 in the presence of MG132...... 85

Figure 27 Truncation of the central region of MDM2 abolishes the interaction

between MDM2 and FOXO1 ...... 86

Figure 28 Immunofluorescence images show the cellular localization of

various MDM2 mutants...... 87

Figure 29 MDM2 promotes the ubiquitination of FOXO1...... 90

Figure 30 MDM2 promotes the ubiquitination of FOXO3A ...... 91

vii Figure 31 The MDM2 ring finger domain is critical for the ubiquitination of

FOXO1 ...... 92

Figure 32 Ubiquitination of FOXO1 requires MDM2 ubiquitin ligase function...... 93

Figure 33 The central region of MDM2 cannot promote the polyubiquitination

of FOXO1 ...... 94

Figure 34 MDM2 promotes FOXO1 polyubiquitination in vitro...... 95

Figure 35 Stably expressed MDM2 in H1299 cells regulates the expression of

downstream targets of FOXO...... 97

Figure 36 MDM2 siRNA increases the expression of FOXO1 target gene

TRAIL ...... 98

Figure 37 MDM2 promotes the cell survival in the presence of FOXO1...... 99

Figure 38 MDM2 protectes cells from FOXO1-induced cell death measured by

Flow Cytometry...... 101

Figure 39 MDM2 increases the transcriptional activity of FOXO1 in a dose-

dependent manner in DU145 cells ...... 104

Figure 40 MDM2 increases the transcriptional activity of FOXO1 in NIH3T3

cells ...... 105

Figure 41 MDM2 enhances the ability of FOXO1 to inhibit cyclin D1 ...... 106

Figure 42 MDM2 increases the transcriptional activity of both wild type FOXO1

and FOXO1 (AAA) mutant in NIH3T3 and DU145 cells ...... 107

Figure 43 P53 inhibits the transcriptional activity of FOXO, in a dose-

dependent manner, but not FOXO1-induced cell death ...... 109

Figure 44 P53 inhibits the transcriptional activity of FOXO in a dose-

dependent manner in LNCaP cells...... 110

Figure 45 MDM2 relieves the repression of FOXO1 activity by p53...... 111

viii Figure 46 MDM2 transiently increases FOXO transcriptional activity, which is

followed by FOXO degradation ...... 112

Figure 47 MG132 relieves p53-induced decrease in the expression of

FOXO3A protein in H1299/V138 cells ...... 113

Figure 48 p53 decreases the protein level of FOXO1 through MDM2...... 114

Figure 49 Knockdown of MDM2 partially relieves p53-induced FOXO3A

downregulation ...... 115

Figure 50 SIRT1 inhibits the transcriptional activity of FOXO...... 117

Figure 51 Nicotinamide treatment increases the FOXO1 transcriptional activity...... 118

Figure 52 SIRT1 sumoylation at Lys 734 is required for FOXO1 deacetylation ...... 119

Figure 53 Mutation of Lys 734 relieves the inhibition of FOXO1 transcriptional

activity by SIRT1...... 120

Figure 54 Genistein increases the expression of FOXO through MDM2

downregulation ...... 122

Figure 55 ARF promotes the MDM2-induced FOXO1 ubiquitination...... 124

Figure 56 A working model shows the functional interaction among FOXO,

p53 and MDM2 ...... 131

ix

List of Tables

Table 1 The FOXO family members in mammals ...... 6

Table 2 Localization and function of different MDM2 mutants...... 133

x

REGULATION OF FOXO STABILITY AND ACTIVITY BY MDM2 E3 LIGASE

WEI FU

ABSTRACT

Members of the forkhead class O (FOXO) transcription factors are tumor suppressors and key molecules that control aging and lifespan. The stability of mammalian FOXO proteins is controlled by proteasome-mediated degradation but general ubiquitin E3 ligases for FOXO factors remain to be defined. The current studies demonstrate that MDM2 bound to FOXO1 and FOXO3A and promoted their ubiquitination and subsequent degradation, a process apparently dependent on FOXO phopshorylation at PKB sites and on the E3 ligase activity of MDM2. The binding occurred between endogenous proteins and was involved the forkhead box of FOXO1 and the region of MDM2 that controls its cellular localization. MDM2 promoted the ubiquitination of FOXO1 in vitro in a cell free system. Knocking down MDM2 by siRNA caused the accumulation of endogenous FOXO3A protein, and enhanced the expression of FOXO target genes. In addition, MDM2 promoted the transcriptional activity of FOXO in a transient transfection system. In cells stably expressing a temperature sensitive mutant p53, activation of p53, by shifting to permissive temperatures led to MDM2 induction and the degradation of endogenous FOXO3A.

These data suggested that MDM2 acts downstream of p53 as an E3 ubiquitin ligase to promote the degradation of mammalian FOXO factors.

xi

INTRODUCTION

1. FOXO Family

1.1. FOX Family

Forkead proteins constitute a family of structurally related transcriptional factors that have been identified in all eukaryotes ranging from to human. Forkhead transcription factors are characterized by the forkhead domain, which is a ~100 amino- acid monomeric DNA binding domain. The three-dimensional structure of forkhead domain consists of two W1 and W2 loops (or wings) and three α helices (Lehmann et al.,

2003). Due to its butterfly-like appearance, the domain is also known as “winged-helix” domain.

The first member of the forkhead family was identified in 1989 as a nuclear homeotic gene involved in the embryonic development of flies (Drosophila melanogaster). To date, over 100 members of the forkhead family have been identified in species ranging from Saccharomyces cerevisiae to (Kaestner et al., 2000). The nomenclature of the forkhead transcription factors has recently been revised. These genes, now termed Fox (after Forkhead box), are divided into 17 subclasses (A to Q),

according to the sequence of their conserved forkhead domains

(http://www.biology.pomona.edu/fox.html).

Comparative genome analyses have shown that the number of forkhead transcription factors appears to have increased during evolution, with a greater number

identified in vertebrates than in invertebrates (Lehmann et al., 2003). The origin and

- 1 - expansion of forkhead genes is positively correlated with eukaryotic complexity because among the for which genome sequences are available, there is a correlation between anatomical complexity and forkhead gene number: 4 FOX genes in S.

cerevisiae, 15 in Caenorhabditis elegans, 20 in Drosophila melanogaster and 43 in

humans (Mazet et al., 2003). The human FOX gene family consists of : FOXA1, FOXA2,

FOXA3, FOXB1, FOXC1, FOXC2, FOXD, FOXD2, FOXD3, FOXD4, FOXD5

(FOXD4L1), FOXD6 (FOXD4L3), FOXE1, FOXE2, FOXE3, FOXF1, FOXF2, FOXG1

(FOXG1B), FOXH1, FOXI1, FOXJ1, FOXJ2, FOXJ3, FOXK1, FOXK2, FOXL1, FOXL2,

FOXM1, FOXN1, FOXN2 (HTLF), FOXN3 (CHES1), FOXN4, FOXN5 (FOXR1), FOXN6

(FOXR2), FOXO1 (FOXO1A, FKHR), FOXO2 (FOXO6), FOXO3 (FOXO3A), FOXO4

(MLLT7), FOXP1, FOXP2, FOXP3, FOXP4, and FOXQ1. Members of FOX subfamilies

A-G, I-L and Q were grouped into class-1 FOX proteins, while members of FOX

subfamilies H and M-P were grouped into class-2 FOX protein. The presence of a C-

terminal basic region within the FOX domain is the common feature of class-1 FOX

protein (Katoh and Katoh, 2004b).

FOX transcriptional regulators play various roles during development, from

organogenesis to language acquistion. Foxb1 is reported to control development of

mammary glands and regions of the central nervous system that regulate the milk

ejection reflex in mice (Kloetzli et al., 2001). FOXC1 and FOXC2 (Kume et al., 2000;

Kume et al., 1998; Wilm et al., 2004), FOXE1 (Aza-Blanc et al., 1993; Ortiz et al., 1997;

Zannini et al., 1997), FOXF1 (Kalinichenko et al., 2001; Kalinichenko et al., 2002),

FOXJ1 (Pelletier et al., 1998) and FOXJ2 (Granadino et al., 2000) play important roles in

the development of the organs, such as thyroid organogenesis, mesonephros, gonad,

liver, gallbladder, lung and intestinal tract. FOXD1 (Hatini et al., 1996), FOXE3 (Blixt et

al., 2007; Valleix et al., 2006) and FOXL2 (De Baere et al., 2001; De Baere et al., 2002)

- 2 - play a critical role in the development of eyes. FOXD3 (Alkhateeb et al., 2005; Guo et al., 2002), FOXD5 (Yu et al., 2002) and FOXJ3 (Landgren and Carlsson, 2004) are involved in neural development. FOXD4 (Minoretti et al., 2007) and FOXP4 (Li et al.,

2004b) are related to heart development and differentiation. The FOXG1 (Pauley et al.,

2006) and FOXI1(Hulander et al., 2003) are required for the development of the mammalian inner ear. FoxH1 (FAST) is a that mediates signaling by transforming growth factor-β, activin, and nodal. FoxH1 / embryos failed to orient the

anterior-posterior (A-P) axis correctly (Yamamoto et al., 2001). FOXJ1 is required for late stages of ciliogenesis (You et al., 2004b). It is essential for nonrandom determination of left-right asymmetry and development of ciliated cells. FOXN4 is necessary and sufficient for commitment to the amacrine cell fate and is nonredundantly required for the amacrine and horizontal cells (Li et al., 2004a). FOXP2 has been implicated in the acquisition of grammatical skills (Hurst et al., 1990; Lai et al., 2001). FOXP3 is the master regulator in the development and function of regulatory T cells and the selected marker for them (Alvarado-Sanchez et al., 2006; Bennett et al., 2001; Brunkow et al.,

2001; Suri-Payer and Fritzsching, 2006). Human FOXN1 (Frank et al., 1999) and

FOXQ1 (Hong et al., 2001) function in hair differentiation. FOXN1 is also required for the growth and differentiation of thymic epithelial cells (Balciunaite et al., 2002).

FOX factors are also very important for cell growth, differentiation and tumorigenesis. FOXA1 binds to the promoters of more than 100 genes associated with metabolic processes, the regulation of signaling and the cell cycle (Carlsson and

Mahlapuu, 2002; Kaestner, 2000; Tomaru et al., 2003). High expression of FOXA1 (Gao et al., 2003; Lin et al., 2002b), FOXN6 (Katoh and Katoh, 2004d), FOXQ1 (Bieller et al.,

2001) and FOXM1(Nakamura et al., 2004) has been reported in various tumors, including lung, esophageal, breast, prostate and pancreatic cancers. Several FOX - 3 - factors are thought of as the candidate tumor suppressor genes. This includes FOXC1

(Zhou et al., 2002), FOXK2 (Li et al., 1992), FOXN5 (Katoh and Katoh, 2004a, c) and

FOXO family (Medema et al., 2000). These factors are reported either to be inactive in cancer or to suppress tumor cell growth when overexpressed. FOXM1 is expressed in proliferating cells and becomes silenced in terminally differentiated cells (Korver et al.,

1997; Yao et al., 1997; Ye et al., 1997). FOXM1 might be beneficial to patients whose lung endothelial-cell barrier has been damaged (Zhao et al., 2006).

FOX factors also regulate chromatin remodeling and transcription. The FOXA3 acts in chromatin remodeling (Kaestner et al., 1994). FOXK1 is essential for regulating cell cycle progression in myogenic progenitor cells (Hawke et al., 2003). FOXL1 is capable of remodeling chromatin higher-order structure and can cause stable and site-

specific changes of chromatin by either creating or removing DNase I hypersensitive sites, resulting in changes the proliferation, differentiation, and positioning of epithelial cells (Kaestner et al., 1997; Yan et al., 2006). FOXN3 binds to Ski-interacting protein

(SKIP, NCoA-62), which is a transcriptional co-regulator known to associate with a repressor complex (Scott and Plon, 2005).

It is believed that FOX factors form a network. For example, FOXG1, a class-1

FOXO member, acts as a repressor of transcriptional activity of the FOXO family (Aoki et al., 2004). FOXA and FOXO factors cooperate in complex regulatory networks of the pancreas and liver (Czech, 2003).

1.2. FOXO Family

FOXO factors are members of the O-subclass of the class-2 FOX family. A unique five amino acid (GDSNS) insertion immediatedly prior to helix H3 within the forkhead domain is characteristic of this subfamily. This insertion is absent in all other

- 4 - FOX transcription factors (Arden, 2006). The first member of FOXO was identified in C. elegans (Caenorhabditis elegans) and named DAF-16 and was shown to regulate dauer formation in C. elegans (Thomas, 1993).

1.2.1. Classification of Human FOXO Factors

In invertebrates there is only one FOXO gene, termed daf-16 in the worm and dFOXO in the fly. In mammals there are four functional FOXO genes, FOXO1, 3A, 4, and 6 (Carter and Brunet, 2007) (Table 1).

The FOXO1 gene is located in 13q14.1. It is the most abundant

FOXO isoform in insulin-responsive tissues such as liver, adipose tissue, pancreatic cells, kidney, spleen and brain (Armoni et al., 2006). Embryos of FOXO1 homozygous mice are smaller and die at embryonic day 10.5 due to several embryonic defects

(Nakae et al., 2002), such as incomplete vascular development (Hosaka et al., 2004).

Analysis of heterozygote mutant mice indicates that FOXO1 is involved in the function of pancreatic β-cells, hepatic glucose metabolism and adipocyte differentiation (Kitamura et al., 2002; Nakae et al., 2002; Nakae et al., 2003).

The FOXO3A gene is located in chromosome 6q21. It is highly expressed in the heart, spleen, lung, kidney, ovary, adipose tissue and brain (Zhu et al., 2004). FOXO3A knockout mice reveal haematological abnormalities, decreased glucose uptake in glucose tolerance tests (Castrillon et al., 2003) and a distinct ovarian phenotype due to premature follicular activation that leads to oocyte death and subsequent depletion of follicles. It has been suggested that FOXO3A acts as a suppressor of follicular activation

(Castrillon et al., 2003; Hosaka et al., 2004). In mature endothelial cells, FOXO1 and

FOXO3A are the most abundant FOXO isoforms. Overexpression of constitutively active

- 5 -

6 FOXO1 or FOXO3A significantly inhibits endothelial cell migration and tube formation in vitro (Potente et al., 2005).

FOXO4 gene is located in chromosome Xq13.1. It is abundant in the lungs, brain, heart and skeletal muscle and kidneys (Nakae et al., 2001). FOXO4 knockout mice show no an obvious abnormalities (Hosaka et al., 2004).

FOXO6 gene is located in 1p34.1. Embryonic expression of FOXO6 is seen predominantly in the developing brain where it is expressed in a specific temporal and spatial pattern. In the adult animal, FOXO6 expression persists in the nucleus accumbens, cingulated cortex, parts of the amygdale, and hippocampus. FOXO6 is also expressed in kidney and thymus (Jacobs et al., 2003). The phenotype of FOXO6- deficient animals is not reported, but its restricted expression pattern suggests that it may play a role in embryologic development of the central nervous system (Hoekman et al., 2006).

1.2.2. Domain Structure of FOXO

From the N-terminus to the C-terminus, the FOXO protein contains a -rich domain, a forkhead domain, NLS (nuclear localization signal), NES (nuclear export signal), a LxxLL motif and an activation domain (Figure 1). The proline rich motif binds to the CH3 region of CBP/p300 and stabilizes the interaction between FOXO factors and

CBP/p300 (van der Heide and Smidt, 2005). The Forkhead domain is responsible for binding to target gene promoters. The central region of FOXO includes the NLS and

NES and accounts for the cellular localization of FOXO. The LxxLL motif is reported to bind to SIRT1 and has an important role in regulating its transcriptional activity (Nakae et al., 2006). The C-terminus of FOXO contains the activation domain which stimulates the activity (Yang et al., 2005). In the nucleus, all FOXO proteins bind to DNA as

- 7 -

PxPxP FOX LxxLL AD 150-260 368-377 461-466 596-655

FOXO1 655aa Nt NLS NES Ct

FOX 148-258 303-327 FOXO3 673aa FOX 88-198 300-308 FOXO4 501aa FOX 78-188 FOXO6 509aa

Figure 1 Structure of human FOXO. Full-length FOXO and known motifs

are presented. PxPxP-proline rich motif; FOX-forkhead domain; LXXLL-L,

leucine; X, any amino acid; AD-activation domain; Nt-amino terminal; NLS-

nuclear localization signal; NES-nuclear export signal; Ct-carboxyl terminal.

The numbers above the drawings denote amino acid numbers.

- 8 - monomers through the FOX domain. The core motif of the consensus recognition site for

FOXO on DNA is GTAAA (C/T) A and it is designated as the DBE for DAF-16 family member-binding element.

1.3. Functions of FOXO Factors

Studies in C. elegans showed that direct activation of DAF-16, or mutation of the

insulin-PI3K (phosphotidylinositol 3 kinase)-PKB (protein kinase B) pathway results in

extension of lifespan, stress resistance and arrest at the dauer diapause stage (Ogg et

al., 1997). Besides cell-autonomous inputs, DAF-16 also responds to environmental

cues such as starvation, heat and oxidative stress. All these stress signals activate DAF-

16, whereas nutrient-rich conditions deactivate it. dFOXO, the unique FOXO homologue

in Drosophila, seems to play similar roles, which are negatively controlled by the insulin-

PI3K-PKB signaling cascade and nutrients (Junger et al., 2003; Kramer et al., 2003;

Puig et al., 2003), although dFOXO-knockout flies are viable and of normal size, they are

more vulnerable to the oxidative stress. These data suggest that dFOXO offers

protection against oxidative stress.

In humans, the pivotal role of FOXO on cell fate decisions depends on the balance between growth factor stimulation versus cellular stress and damage. Both

circumstantial and direct evidences implicate a role of FOXO factors in cancer. The first three members of FOXO were found at chromosomal translocations in tumors. The

FOXO1 gene is fused to PAX3 or PAX7 gene in rhabdomyosarcoma (Galili et al., 1993;

Sorensen et al., 2002). The FOXO3A gene is fused to MLL gene in secondary acute myeloblastic leukemia (Hillion et al., 1997). The FOXO4 gene is fused to MLL gene in acute lymphocytic leukemia (Parry et al., 1994). Nuclear exclusion of FOXO3A in primary breast tumors correlates with PI3K activation and poor survival of the patients

(Hu et al., 2004). FOXO factors reduce tumorigenicity in nude mice (Hu et al., 2004; - 9 - Ramaswamy et al., 2002). Furthermore, FOXO proteins interact with many oncogenes

such as β-catenin (Essers et al., 2005) or tumor suppressors such as p53 (Brunet et al.,

2004).

The physiological importance of FOXO transcription factors is diverse as revealed by loss and gain of function experiments in transgenic and knockout mice. All lines of evidence point to FOXO factors as central regulators of metabolism, aging, proliferation and differentiation (Hosaka et al., 2004). Since a wide range of human diseases, including cancer, have striking aging-dependent onset, FOXO factors are believed to serve as molecular links between longevity and tumor suppression (Greer and Brunet, 2005).

1.3.1. FOXO and Cell Cycle Checkpoint and DNA Repair

Mammalian cells have evolved an intricate defense network to maintain genomic integrity by preventing the fixation of permanent damage from endogenous and exogenous mutagens. Cell cycle checkpoints, a major genomic surveillance mechanism acting at the G1/S and G2/M boundaries, are regulated in response to DNA damage

(Hartwell and Weinert, 1989). Defects in these steps may result in a mutated phenotype

that is associated with tumorigenesis.

1.3.1.1. FOXO and G1/S Checkpoint

p21 is a cyclin- dependent kinase inhibitor. Loss of p21 expression has been

described as a poor prognostic factor and as an independent predictor of bladder cancer

progression in muscle invasive cancer (Stein et al., 1998). p27 is a protein that binds to

cyclins and cdk to block the entry into S phase. Loss of p27 expression has been shown

to be associated with aggressive behavior in a variety of human epithelial tumors,

including prostate (Macri and Loda, 1998) and breast (Catzavelos et al., 1997; Gillett et

- 10 - al., 1999; Tan et al., 1997; Wu et al., 1999) cancers. p130 belongs to the RB family. A study in mice showed that p130 can induce cell cycle arrest (Classon et al., 2000).

Mammalian cyclins are classified into 12 different types, from cyclins A to I, based on structural similarity, function period in the cell division cycle, and regulated protein expression. cyclin D1 is a key regulator of the G1 phase of the cell division cycle where it binds to and activates the cyclin-dependent kinases CDK4 and CDK6. In the cells that have the capacity to divide, the main effect of the expression of active forms of FOXO family members is to promote cell cycle arrest at the G1/S boundary. FOXO factors play a major role in G1 arrest by upregulating cell cycle inhibitors such as p21 (Hauck et al.,

2007; Lawlor and Rotwein, 2000), p27 (Medema et al., 2000; Stahl et al., 2002) and p130 (Chen et al., 2006) and by repressing cell cycles activators cyclin D1 and D2

(Schmidt et al., 2002).

1.3.1.2. FOXO and G2/M Checkpoint and DNA Repair

GADD45 is growth arrest and DNA damage-inducible protein 45. It was identified on the basis of its rapid transcriptional induction after UV irradiation (Fornace et al.,

1989) and can induce G2/M arrest (Wang et al., 1999). Induction of GADD45 is also observed after several types of pathological stimuli including various environmental stresses: hypoxia, irradiation, genotoxic drug exposure and growth-factor withdrawal

(Papathanasiou et al., 1991).Three G-type cyclins have been identified: cyclins G1, G2, and I. All three are expressed in terminally differentiated cells, act as cell cycle inhibitors in certain cell types and may induce cell cycle arrest (Martinez-Gac et al., 2004).

Microarray analysis led to the identification of GADD45 and cyclin G2 as the downstream target genes of FOXO3A for G2/M arrest and DNA repair (Tran et al.,

2002).

- 11 - In vivo experiment showed that FOXO3A and FOXO4 activate GADD45 promoter through direct interaction with the DBE. Oxidative stress activates the GADD45 promoter in a FOXO-dependent manner, resulting in increased level of GADD45 mRNA and protein as well as G2 arrest (Furukawa-Hibi et al., 2002). In response to glucose starvation, FOXO1 also affects GADD45 mRNA level in islets (Martinez et al., 2006).

Cyclin G2 is expressed in various amounts during the cell cycle in lymphocytes

(Horne et al., 1997; Horne et al., 1996). Ectopic expression of cyclin G2 inhibits cell

cycle progression (Bennin et al., 2002). FOXO3A interacts with cyclin G2 in mouse

fibroblasts. Active forms of FOXO3A increase cyclin G2 mRNA levels by activating the

cyclin G2 transactivation through interaction with the DBE in its promoter (Martinez-Gac

et al., 2004).

One mechanism by which cells protect themselves against stress is by repairing

the damage to their DNA and proteins that occurs upon exposure to environmental

stress. Furthermore, this capacity to repair DNA damage is closely correlated with an

increased longevity in mammals (Kirkwood and Austad, 2000). Gene array analysis

showed that FOXO3A is involved in nuclear excision DNA repair by modulating the

expression of GADD45 and DDB (damaged DNA binding) protein (Tran et al., 2002).

1.3.2. FOXO and Apoptosis

FOXO factors have been shown to mediate apoptosis by activating proapoptotic genes in a variety of cells. FOXO promotes cell death through these downstream targets: TRAIL (Tumor necrosis-related apoptosis-inducing ligand), FasL (Fas Ligand)

(Brunet et al., 1999), Bim (Stahl et al., 2002; Sunters et al., 2003; Urbich et al., 2005) and PUMA (You et al., 2006a).

TRAIL is a member of the TNF family of cytokines. It induces apoptosis via death receptors (DR4 and DR5) in a wide variety of tumor cells but not in normal cells (Suliman - 12 - et al., 2001). TRAIL is a direct target of FOXO3A. FOXO3A response element is located in TRAIL promoter spanning nucleotides -138 to -121. Decreased activity of FOXO3A and FOXO1 in prostate cancers resulting from loss of PTEN leads to a decrease in

TRAIL expression that can contribute to increased survival of the tumor cells (Modur et al., 2002).

FasL is a type II transmembrane protein. It functions as a homotrimer, because it trimerizes Fas , which spans the membrane of the "target" cell. This trimerization usually leads to apoptosis.There are three putative FOXO binding elements in the FasL promoter. In the human leukemia T cell line Jurkat, FOXO3A induces

apoptosis by activating FasL expression and the Fas-FasL apoptotic pathway

(Yamamura et al., 2006).

Bim is a BH3-only Bcl2 family member. Bim (also known as Bcl2l11) provokes

apoptosis (O'Connor et al., 1998). There are two conserved FOXO binding sites in the

Bim promoter: one at position -204 relative to the transcription start site (bim1) and one

at the boundary between exon 1 and the first intro. FOXO3A directly activates the bim

promoter via the two conserved FOXO binding sites (Gilley et al., 2003).

PUMA is an essential mediator of p53-dependent and -independent apoptosis in

vivo. Promoter analysis identifies that there is a potential consensus FOXO-responsive

element in intron 1 of PUMA which is conserved in humans and mice. FOXO3A up- regulates PUMA transcription in response to cytokine or growth factor deprivation (You

et al., 2006a).

1.3.3. FOXO and Atrophy

Atrophy is the partial or complete decompositon of a part of the body. Causes of

atrophy include poor nourishment, poor circulation, loss of hormonal support, loss of

nerve supply to the target organ, disuse or lack of exercise or disease intrinsic to the - 13 - tissue itself. Hormone and nerve inputs that maintain an organ or body part are referred to as trophic. Atrophy is a general physiological process of reabsorption and breakdown of tissues, involving apoptosis on a cellular level. When it occurs as a result of disease or loss of trophic support due to other disease, it is termed pathological atrophy, although it can be a part of normal body development and homeostasis as well.

Skeletal muscle atrophy is a debilitating response to fasting, disuse, cancer, and other systemic diseases. FOXO3A acts on the atrogin-1 promoter to cause atrogin-1 transcription and dramatic atrophy of myotubes and muscle fibers. When FOXO activation is blocked by a dominant-negative construct in myotubes or by RNAi in mouse muscles in vivo, atrogin-1 induction is prevented during starvation and atrophy of myotubes induced by glucocorticoids (Sandri et al., 2004).

In fully differentiated skeletal and cardiac muscle cells, expression of a constitutively active form of FOXO3A causes atrophy. The atrophy is not due to apoptosis. Instead, it is due to a decrease in cell size.

1.3.4. FOXO and ROS Detoxification in Stem Cells

FOXO proteins have been reported to allow detoxification of ROS (reactivie oxygen species) by upregulating free radical scavenging enzymes, including MnSOD

(manganese superoxide dismutase) and catalase (Storz, 2006).

FOXO3A protects quiescent cells from oxidative stress by directly increasing

MnSOD mRNA and protein levels. This increase in protection from reactive oxygen species antagonizes the apoptosis caused by glucose deprivation. Increased resistance to oxidative stress is also associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in C. elegans, but the model also can be extendable to mammalian systems (Kops et al., 2002).

- 14 - Stem cells are primal cells found in all multi-cellular organisms. They have two important characteristics that distinguish them from other types of cells. First, they are unspecialized cells that renew themselves for long periods through cell division. The second characteristic is that under certain physiologic or experimental conditions they can be induced to become cells with special functions such as the beating cells of the heart or the insulin-producing cells of the pancreas. Regulation of oxidative stress in the

HSC (hematopoietic stem cell) compartment is critical for the maintenance of HSC self- renewal. FOXO-dependent signaling is required for the long-term regenerative potential of the HSC compartment through regulation of HSC responses to physiologic oxidative stress, quiescence and survival. Further analysis of ROS levels in FOXO knockout mice showed that FOXOs affect HSC integrity by regulating ROS. Myeloid progenitor cells isolated from FOXO1/3/4 conditional knockout animals show decreased ROS (Tothova et al., 2007).

1.3.5. FOXO and Tissue Differentiation

Cell and tissue differentiation process systematically bases on a number of gene expressions that commence successively along with the passage of time. In differentiating cells, FOXO factors have been implicated in inhibiting and promoting differentiation, depending on the cell types and different FOXO isoforms. In adipocytes,

FOXO1 is induced in the early stages of adipocyte differentiation but its activation is delayed until the end of the clonal expansion phase. Constitutively active FOXO1 prevents the differentiation of preadipocytes, while dominant-negative FOXO1 restores adipocyte differentiation of fibroblasts from insulin receptor-deficient mice. FOXO1 directly inhibits the differentiation through upregulation of p21 (Nakae et al., 2003).

Hematopoiesis, or blood cell formation, is the process in which a limited set of hematopoietic stem cells is able to give rise to all types of functional blood cells via - 15 - commitment to specific hematopoietic lineages. FOXO3A can promote erythroid differentiation through BTG1 (B-cell translocation gene 1). BTG1, in turn, modulates necessary for erythroid differentiation (Bakker et al., 2004).

Furthermore, recent experiments demonstrated that FOXO3A directly binds and represses the transcription of the ID1 (inhibitor of differentiation 1), a suppressor of erythroid differentiation, through the recruitment of an HDAC1 ( deacetylase 1)– mSin3a complex (Lam et al., 2006) .

1.3.6. FOXO and Glucose and Energy Metabolism

FOXO factors play an important role in upregulating genes that control glucose metabolism. They upregulate G6Pase (glucose-6-phosphatase) which is responsible for converting glucose-6-phosphate to glucose (Nakae et al., 2001; Onuma et al., 2006) and

PEPCK (phosphoenolpyruvate carboxykinase) which converts oxaloacetate to phosphoenolpyruvate (Samuel et al., 2006). In the absence of insulin, FOXO1 binds to

IRE (insulin response elements) in G6Pase and PEPCK promoters and stimulates their promoter activity. However, in the presence of insulin, FOXO1 undergoes phosphorylation and is exported out of the nucleus, ceasing its transcriptional activity

(Kitamura et al., 2002). In liver and muscle, FOXO proteins can stimulate PDK4 (

pyruvate dehydrogenase kinase-4) expression, which limits oxidative metabolism of glucose and conserves glucose for utilization in other tissues (Furuyama et al., 2003).

FOXOs are also involved in lipid metabolism through the regulation of AdipoR1/2

(Tsuchida et al., 2004), LPL (lipoprotein lipase) (Kamei et al., 2003), HMGCS2

(mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase) (Nadal et al., 2002) and SCP

(sterol carrier protein) (Dansen et al., 2004) . Adiponectin/Acrp30 is a hormone secreted by adipocytes, which acts as an antidiabetic and antiatherogenic

adipokine. AdipoR1 and -R2 serve as the receptors for adiponectin and mediate - 16 - increased fatty acid oxidation and glucose uptake by adiponectin. Mouse study shows

that insulin reduces the expression of AdipoR1/R2 via the PI3K/FOXO1-dependent

pathway in vitro (Tothova et al., 2007). FOXO proteins also stimulate the expression of

both PDK4 (Furuyama et al., 2003; Kwon et al., 2004), which limits the flux of pyruvate

through the Krebs cycle, and the expression of LPL (Kamei et al., 2003). HMGCS2 is a

key enzyme controling ketogenesis. Deletion analysis showed that there is a FOXO3A-

responsive sequence AAAAATA located 211 bp upstream of the HMGCS2 gene

transcription start site. FOXO3A can stimulate transcription of HMGCS2 and this

stimulation is repressed by insulin (Nadal et al., 2002). The SCP (sterol carrier protein)

gene encodes two proteins, SCPx and SCP2, which are independently regulated by

separate promoters. SCPx has been shown to be the thiolase involved in the breakdown

of branched-chain fatty acids and in the biosynthesis of bile acids. Both SCPx and SCP2

are upregulated by FOXO3A (Dansen et al., 2004).

1.3.7. FOXO and Longevity

A number of diseases, including cancer, type 2 diabetes and neurodegenerative

disorders have a striking age-dependent onset. A number of studies now support the

hypothesis that longevity could be affected by simple changes in the environment.

Organismal longevity also has a genetic component. A pivotal breakthrough in

identifying genes involved in longevity came from studies in C. elegans. Some worm

mutants can live two to three times longer than wild type worms (Kenyon et al., 1993).

These long-living worms turned out to have mutations in the insulin receptor gene

(Kimura et al., 1997). Remarkably, flies and mice that have mutations in the insulin or

the IGF-1 (insulin like growth factor-1) receptor genes are also long-lived (Bluher et al.,

2003; Holzenberger et al., 2003; Tatar et al., 2001). These results indicate that insulin

and IGF-1 regulate longevity in a conserved manner throughout species. - 17 - A link between FOXO factors and aging was also initially observed in invertebrates. In C. elegans, DAF-16 regulates lifespan (Baumeister et al., 2006). In

Drosophila melanogaster, dFOXO regulates age-linked decline of cardiac function and

longevity (Giannakou et al., 2004; Giannakou and Partridge, 2004; Hwangbo et al.,

2004; Wessells et al., 2004).

But so far, there is no direct evidence to link FOXO factors and longevity in

mammals. Since FOXO factors are involved in cellular resistance to stress and promote

ROS detoxification, DNA repair, and cell cycle arrest to allow time for the repair process,

they are projected to reduce detrimental effects of aging in mammals (Glauser and

Schlegel, 2007). Thus, a role of FOXO factors in longevity of mammals is expected.

1.4. Posttranslational Modifications of FOXO Proteins

As described above, FOXO proteins have diverse cellular functions by acting as

transcription factors. The activity of FOXO proteins can be regulated by posttranslational

modifications including phosphorylation, acetylation and ubiquitination.

1.4.1. Phosporylation of FOXO

Phosphorylation is a process wherein phosphate groups are added to proteins by

protein kinases or removed from proteins by protein phosphatases. The addition or

removal of phosphate groups dramatically alters protein function and leads to a myriad

of biological responses. Phosphorylation is shown to play a major role in the regulation

of FOXO cellular localization, transcription activity and protein stability, and is mediated

mainly by PKB.

1.4.1.1. PKB and FOXO

PKB is a / kinase. It plays a pivotal role in cell survival and

proliferation through a number of downstream effectors. All FOXO family members, - 18 - except FOXO6, contain three PKB phosphorylation sites (RXRXX(S/T)X). DAF-16, the

C. elegans FOXO, contains four putative PKB recognition motifs. Two are located at end of the N-terminal & C-terminal of DAF-16. The other two sites are located in the forkhead domain and overlap. Disrupting PKB-consensus phosphorylation sites in DAF-16 causes nuclear accumulation in wild-type animals but has little effect on lifespan (Lin et al.,

2001; Van Der Heide et al., 2004).

PKB is shown to phosphorylate human FOXO family members on the three PKB motifs: an N-terminal threonine (Thr32), a forkhead box serine (Ser253), and a more C- terminal serine (Ser315). Thr32 is preferentially phosphorylated by PKB. This phosphorylation is shown to inhibit p300 binding to FOXO3A, resulting in the recruitment of RanGTPase and CRM1 protein to transport FOXO3A to cytoplasm and to bind the 14-

3-3 (Mahmud et al., 2002). Mutation of Ser253 to a neutral residue, such as alanine (S253A), inhibits further phosphorylation of FOXO3A in other regions of the proteins by PKB and other kinases. This suggests that this site is necessary to prime other sites for phosphorylation. The FOXO3A S253A mutant is constitutively nuclear under different conditions including PKB activation. Therefore, the S253 is called the

“gatekeeper site”. This site is believed to inhibit DNA binding, to change the conformation and to inhibit nuclear re-import (Brownawell et al., 2001; Brunet et al.,

1999; Brunet et al., 2001; Woods and Rena, 2002). Phosphorylation of Ser315 is shown to enhance nuclear export (Brunet et al., 2001; Rena et al., 2002). All FOXO proteins have been shown to require the consensus N-terminal PKB site and the PKB site located in the forkhead domain in order to translocate from nucleus to

(Brownawell et al., 2001).

Interestingly, the regulation of FOXO6 is different from those of FOXO1,

FOXO3A and FOXO4. FOXO6 only contains two PKB/SGK regulatory phosphorylation

- 19 - sites (Thr26 and Ser184 in mouse FOXO6) (Jacobs et al., 2003). Unlike the other FOXO isoforms, FOXO6 is mostly nuclear. However, FOXO6 phosphorylation at Thr26 and

Ser184 appears to decrease its transcriptional activity (van der Heide and Smidt, 2005), which is similar to the effect of PKB on other FOXO factors transcriptional activity.

1.4.1.2. SGK and FOXO

SGKs (serum- and glucocorticoid-induced kinases) belong to a new family of serine/threonine kinases that are regulated at both the transcriptional and posttranslational levels by external stimulation. SGK mediates the biological effects of

PI3K in parallel with PKB. Although SGK is closely related to PKB, SGK and PKB display unique features. First, SGK protein expression is induced by extracellular stimuli, but PKB might not be. Second, SGK does not have a pleckstrin homology domain and appears not to be recruited to the plasma membrane prior to its activation. Third, the consensus sequence that is phosphorylated by SGK is not identical to the site phosphorylated by PKB. Fourth, SGK, in contrast to PKB, is capable of phosphorylating

peptide substrates that do not have a bulky hydrophobic amino acid immediately C-

terminal to the phosphate-acceptor site (Kobayashi and Cohen, 1999).

Activated SGK promotes cell survival in part by phosphorylating and inactivating

FOXO3A. In the presence of DNA damage, caused by UV and irradiation treatment,

SGK is induced in a p53-dependent manner, leading to the phosphorylation of FOXO3A

(You et al., 2004a). SGK, like PKB, phosphorylates FOXO3A, thereby causing FOXO3A

translocation from the nucleus to the cytoplasm and inhibition of FOXO3A-dependent

transcription. However, SGK and PKB, when expressed at physiological levels, display

differences with respect to the efficacy with which they phosphorylate the regulatory

sites on FOXO3A. Specifically, Thr32 is phosphorylated by both protein kinases, but

SGK prefers Ser315 whereas PKB favors Ser253 (Brunet et al., 2001). - 20 - 1.4.1.3. IκB Kinase and FOXO3A

The IKK (IκB kinase) signaling pathway is a key survival and antiapoptotic mechanism, aberrant expression of IKK has been implicated in constitutive activation of

NF-κB in human breast cancer cell lines and primary tumors. In breast cancer, IKK physically interacts with, phosphorylates, and inhibits FOXO3A independently of PKB.

Cytoplasmic FOXO3A correlates with expression of IKKβ or phospho-PKB in many tumors and associates with poor survival in breast cancer (Hu et al., 2004).

1.4.1.4. CK1 and FOXO

CK1 (Casein Kinase 1) is a serine/threonine protein kinase (Hathaway and

Traugh, 1979) that phosphorylates FOXO1 at Ser322 and Ser325 following the phosphorylation of Ser319 by PKB or SGK. Multi-site phosphorylation of the region containing Ser319, Ser322, Ser325 and Ser329 provides a signal for the nuclear exclusion of FOXO1 (Rena et al., 2002).

1.4.1.5. CDK2 and FOXO

CDK2 phosphorylates FOXO1 at serine-249 (Ser249) in vitro and in vivo.

Phosphorylation of FOXO1 at Ser249 results in cytoplasmic localization of FOXO1 and

the inhibition of its transcriptional activity. This phosphorylation was abrogated upon DNA

damage through the cell cycle checkpoint pathway and was dependent on the protein

kinases Chk1 and Chk2 (Huang et al., 2006). Functional interaction between CDK2 and

FOXO1 provides a mechanism that regulates apoptotic cell death after double strand

DNA breakage.

1.4.1.6. JNK and FOXO

Besides IIS (insulin/IGF signaling) pathway, lifespan can also be increased by activating the stress-responsive JNK (Jun-N-terminal kinase) pathway. In Drosophila, - 21 - JNK requires FOXO to extend lifespan. JNK antagonizes IIS, causes nuclear localization of FOXO and induces the expression of FOXO targets including the growth control and stress defense genes. JNK and FOXO also restrict IIS activity systemically by repressing

IIS ligand expression in neuroendocrine cells (Wang et al., 2005).

In humans, an increase in ROS levels induces activation of the small GTPase

Ral, which in turn leads to the phosphorylation and activation of the stress kinase JNK.

Active JNK induces the phosphorylation of Thr447 and Thr451 on FOXO4.

Phosphorylation of these residues is essential for FOXO4 transcriptional activity.

Consistent with this, H2O2 treatment increases FOXO transcriptional activity and

translocation of FOXO4 from the cytoplasm to the nucleus and activation of the

transcription factor. Activation of FOXO4 through Thr447/451 phosphorylation can

induce transcription of MnSOD and catalase, leading to a decrease in ROS levels. Thus,

activation of FOXO4 by oxidative stress is part of a negative feedback loop to reduce the

levels of oxidative stress in a cell and to prevent damage to DNA, lipids and proteins

(Essers et al., 2004).

1.4.1.7. MST1 and FOXO3A

MST1 is a serine/threonine protein kinase that mediates cell death induced by

oxidative stress in primary mammalian neurons through direct activation of FOXO

transcription factors. MST1 phosphorylates FOXO proteins at a conserved site within the

FOX domain (Ser207) disrupting their interaction with 14-3-3 proteins, promoting FOXO

nuclear translocation, and thereby inducing cell death in neurons under stress conditions

such as hydrogen peroxide treatment. Knockdown of the C. elegans MST1 ortholog

CST-1 shortens lifespan and accelerates tissue aging, while overexpression of cst-1

promotes lifespan and delays aging. The cst-1-induced lifespan extension requires daf-

16 (Lehtinen et al., 2006). - 22 - 1.4.2. Acetylation and Deacetylation of FOXO

1.4.2.1. Acetylation and Deacetylation

Protein acetylation is a post-translational modification that transfers an acetyl group to residues. The best example is histone acetylation, which “opens” chromatin’s structure and activates transcription. Deacetylation, on the other hand, induces closed chromatin structure and repression of gene expression (Cohen and Yao,

2004). Protein acetylation is catalyzed by a group of acetyltranferases with different specificities and target consensus sites. CBP/p300 (calcium response element-binding

(CREB)-binding protein), PCAF (p300/CBP-associated factor), members of p160 family,

Tip60, and TAFII250 are the main acetyltransferases involved in histone regulation and transcription factor acetylation (http://www.chromatin.us/hatdesc.html). Proteins acetylation is reversed by HDACs (histone deacetylases). There are three classes of

HDACs. Class I includes HDAC1, 2, 3, and 8. Class II includes HDAC4, 5, 6, 7, 9 and 10 which show high homology to HAD1 yeast deacetylase. There is one HDAC (HDAC11) that shares homology with both class I and II HDACs. Class III HDACs (SIRTs) have an absolute requirement for NAD in vivo and in vitro (de Ruijter et al., 2003; Imai et al.,

2000).

1.4.2.2. Acetylation of FOXO

1.4.2.2.1. P300 and CBP

p300 and CBP are large nuclear proteins encoded by two distinct genes. p300/CBP have been implicated in numerous disease processes, including several forms of cancer, cardiac hypertrophy and Huntington’s disease (Bandyopadhyay et al.,

2002; Borrow et al., 1996; Deguchi et al., 2003; Gayther et al., 2000; Gusterson et al.,

2003; Muraoka et al., 1996; Ross et al., 2002). - 23 - The relative low abundance of p300 is rate-limiting in coactivation and corepression of many transcription factors. Thus, p300 serves to integrate diverse signaling pathways involved in metabolism, cellular differentiation and p53-mediated apoptosis processes (Yao et al., 1998). Orchestration of these activities by p300 involves both a scaffolding function to tether transcription factors to target promoters and its enzymatic activity as a HAT (histone acetyl transferase) (Bannister and Kouzarides,

1996; Ogryzko et al., 1996).

In addition to , several other non-histone proteins including transcription factors are also acetylated by p300 (Goodman and Smolik, 2000), including p53, (Wang et al., 2001) and MyoD (Polesskaya et al., 2000). The activity of p300 itself is also subjected to regulation via a number of post-translational modification including phosphorylation, methylation, sumoylation and acetylation

(Banerjee et al., 1994; Chevillard-Briet et al., 2002; Girdwood et al., 2003; Thompson et al., 2004; Yaciuk and Moran, 1991; Yadav et al., 2003).

In terms of differentiation, p300 and CBP appear to have numerous functions.

There is embryonic lethality of mice nullizygous for p300 (with defects in neurulation and heart development) as well as in mice double heterozygous for p300 and CBP. Animals which are nullizygous for p300 died between days 9 and 11.5 of gestation and showed defects in neurulation, cell proliferation and heart development. Cells derived from p300- deficient embryos displayed specific transcriptional defects and proliferated poorly.

Surprisingly, p300 heterozygotes also manifested considerable embryonic lethality.

Moreover, double heterozygosity for p300 and CBP was invariably associated with embryonic death. Thus, mouse development is exquisitely sensitive to the overall gene dosage of p300 and CBP (Yao et al., 1998). Further study in the mouse oocytes within primordial follicles finds that p300 and CBP enter into the oocyte nucleus at different

- 24 - stages of oocyte growth. From the two-cell stage to the blastocyst stage in the pre- implantation mouse embryos, the localizations of p300 and CBP are different, which provide the information that p300 and CBP have different functions in early mouse embryogenesis (Kwok et al., 2006).

1.4.2.2.2. P300/CBP and FOXO Acetylation

Using immunoprecipitation, an interaction between FOXO1, FOXO4, and

FOXO3A and CBP as well as between FOXO3A and p300 was found in vivo and in vitro

(Brunet et al., 2004; Daitoku et al., 2004; Fukuoka et al., 2003; Motta et al., 2004; van der Horst et al., 2004).

CBP and p300 play different roles in the regulation of FOXO1 and FOXO4.

Mutational analysis show that FOXO1 and FOXO4 interact with CBP via their C-terminal domain, whereas the N-terminal CH1 domain of CBP is required to bind FOXO1 and

FOXO4. CBP binds and acetylates FOXO1 at the K (lysine) 242, K245, and K262

residues. Acetylation at these residues by CBP attenuates FOXO1’s ability to bind cognate DNA sequence and promotes the PKB-dependent phosphorylation of FOXO1

(Matsuzaki et al., 2005). CBP interacts with FOXO4 and acetylates it at K186, K189, and

K408. Acetylation by CBP inhibited the transcriptional activity of FOXO4 (Daitoku et al.,

2004; Fukuoka et al., 2003; van der Horst et al., 2004). However, in transient transfection experiments acetylation by p300 in the C-terminal region of FOXO1 showed that it potently stimulates FOXO1-induced transcription of IGF-binding protein-1. The intrinsic acetyltransferase activity of p300 is required for both activities (Perrot and

Rechler, 2005).

As for FOXO3A, p300 and CBP can bind and acetylate it both in vivo and in vitro, but two different studies show some discrepancies. One study shows that FOXO3A binds p300/CBP at the N-terminus masking the first 52 amino acids at the N-terminus - 25 - under serum starvation conditions (Mahmud et al., 2002). Another study shows that

CBP/p300 binds the C-terminus and forkhead domains of FOXO3A, leading to acetylation at lysine residues K242, K259, K271, K290, and K569 (Brunet et al., 2004;

Mahmud et al., 2002). Overall, acetylation by p300 stimulates the transcriptional activity

of FOXO3A (Motta et al., 2004).

1.4.2.3. Deacetylation of FOXO by HDAC

1.4.2.3.1. SIRT1

The SIRT1 gene is located in chromosome 10q21.3. It contains 9 exons

(NC 000010). The 4,107 bp human Sirt1 mRNA has an open reading frame of 2,244 bp

and encodes a 747 aa protein with a predictive molecular weight of 81.7 kDa (kilodalton)

(Voelter-Mahlknecht and Mahlknecht, 2006). It belongs to the family of mammalian sirtuins. The founding member of the sirtuin protein family was the silent information regulator 2 (Sir2) of S. cervisiae (Saccharomyces cervisiae). Sirtuin proteins are highly

conserved from yeast to humans. In yeast, worms and flies, expression of Sir2 extends

longevity (Kaeberlein et al., 1999; Rogina and Helfand, 2004; Tissenbaum and

Guarente, 2001; Wood et al., 2004). In S. cervisiae, there are five sirtuin homologs,

Hst1-4 and Sir2. In mammals, there are seven sirtuins, SIRT1-7. SIRT1 is the closest

homolog of Sir2, plays important roles in diverse cellular processes including

transcriptional silencing, rDNA recombination, glucose metabolism and energy

homeostasis, DNA repair and cell survival. Due to its dependency on NAD+, the activity

of SIRT1 is regulated by NAD+/NADH ratio and thus sensitive to the redox status and

celluar metabolism. Similar to Sir2, SIRT1 is potentially a nutrient sensor that regulates

the lifespan of mammals in response to caloric restrication or nutrient starvation.

- 26 - Several compounds have been shown to inhibit or activate the deacetylase activity of the Sir2 family. One inhibitor, splitomicin, is a cell-permeable lactone derived from β-naphthol; it inhibits the NAD+-dependent deacetylase activity of Sir2 in vitro

(Bedalov et al., 2001). Another inhibitor, nicotinamide, a product of the Sir2 deacetylation reaction, is an inhibitor of Sir2 activity both in vivo and in vitro. Nicotinamide has been shown to inhibit a Sir2 homolog, SIRT1, a negative p53 regulator, promoting p53- dependent apoptosis in mammalian cells (Luo et al., 2001; Vaziri et al., 2001). The most potent activator was resveratrol, a polyphenol found in red wine, which is implicated in a number of health benefits. In human cells, treatment with a low concentration of resveratrol increased cell survival following DNA damage. Moreover, low resveratrol concentrations decreased the acetylation of p53 at lysine residue 382, a known SIRT1 substrate; however, high resveratrol concentrations caused the opposite (Howitz et al.,

2003).

By deacetylating and inactivating the substrates such as p53 (Langley et al.,

2002; Vaziri et al., 2001), NFκB (nuclear factor-κB), FOXO (Brunet et al., 2004; Daitoku et al., 2004; Motta et al., 2004; van der Horst et al., 2004), Ku70 (Jeong et al., 2007),

PPARγ (Peroxisome proliferator-activated receptor γ) (Picard et al., 2004), PGC-1α

(Balaban et al., 2005), AceCS2 (acetyl-CoA synthetase 2), p300 (Bouras et al., 2005), α- , and HES1 and HEY2 (basic helix–loop–helix transcriptional repressors) (Takata and Ishikawa, 2003), SIRT1 plays important roles in regulating various cellular processes, including stress response (Vaziri et al., 2001), embryogenesis (McBurney et al., 2003), metabolism (Leibiger and Berggren, 2006), calorie restriction (Brunet et al.,

2004), neuronal cell survival (Anekonda and Reddy, 2006; Bordone et al., 2006), insulin secretion (Bordone et al., 2006) and aging (Chua et al., 2005).

- 27 - 1.4.2.3.2. SIRT1 and FOXO Deacetylation

In mammalian cells, SIRT1 interacts with all the FOXO factors. Co-

immuoprecipitation experiments showed that SIRT1 interacts with FOXO1 in vivo.

Mutation analysis showed that the LxxLL motif in FOXO1 (amino acids 459-463) is

critical for the interaction with SIRT1. Mutagenesis of the LxxLL motif eliminates FOXO1

interaction with SIRT1, sustains the acetylated state of FOXO1 and makes FOXO1

insensitive to nicotinamide and resveratrol (Nakae et al., 2006). Yeast two hybridization

experiment showed that FHL2 (Four and a half LIM 2), which interacts with the N-

terminal of FOXO1 in vivo and in vitro, can enhance the interaction of FOXO1 and

SIRT1 (Yang et al., 2005).

Mammalian SIRT1 has dual effects on FOXO3A and FOXO4. SIRT1 deacetylates

FOXO3A, attenuating FOXO-induced apoptosis but potentiates FOXO-induced cell-cycle

arrest (Motta et al., 2004). SIRT1 and FOXO3A form a complex in cells in response to

oxidative stress, and SIRT1 deacetylates FOXO3A in vitro and in intact cells. The sites of

FOXO3A that appear to be primarily deacetylated by SIRT1 are K242, K245, and K262

(Daitoku et al., 2004). The SIRT1 and FOXO3A interaction requires PKB

phosphorylation of FOXO3A because constitutive FOXO3A, in which three PKB

phosphorylation sites were mutated to alanine, failed to bind SIRT1 under any stress

condition (Brunet et al., 2004; Motta et al., 2004). SIRT1 has a dual effect on FOXO3A function: It increases FOXO3A's ability to induce cell cycle arrest and resistance to

oxidative stress, but inhibits FOXO3A's ability to induce cell death (Brunet et al., 2004).

In the case of FOXO4, acetylation by CBP inhibits its transcriptional activity.

SIRT1- mediated deacetylation precludes FOXO4 inhibition through acetylation andthereby prolongs FOXO4-dependent transcription of stress-regulating genes. A

FOXO4 study revealed a molecular mechanism whereby SIRT1 can promote cellular

- 28 - K245 K248 K259 K265 K274 K294 FOXO1 K242 K245 K259 K262 K271 K290 K569 FOXO3 K182 K185 K199 K211 K233 K403 FOXO4 K173 K176 K190 K202 K229 FOXO6

1 150 A A A A A A 368 377 461 466 596 655

FOX FOXO1 P P P P P P P P P P CDK2

T24 S249 S256 S319 S322 S325 S329 FOXO1 T32 S253 S315 S318 S321 S325 S644 FOXO3 T28 S193 S258 S261 S264 S268 T447 T451 FOXO4 T26 S184 FOXO6

PKB/SGK CK1 DYRK JNK IKKβ 150 260 SMAD3/4 1 150 208 655 P300/CBP P300/CBP 1 150 260 368-377 461-466 FHL2 FOXG1 Crm1 SIRT1 401 652 P53 SHP

24 256 350 655 ∗ 14-3-3 ∗ AR

Figure 2 The regulation of FOXO. Full-length FOXO and known

phosphorylation sites and acetylation sites are presented. K-lysine; T-threonine;

S-serine; T-; CDK2-cyclin-dependent kinase 2; PKB- protein Kinase B;

SGK- serum and glucocorticoid responsive kinase; CK1- casine kinase 1;DYRK-

dual-specificity YAK-1-related kinase; JNK- c-Jun N-terminal kinase; IKKβ-IκB

kinase β. The numbers above the drawings denote amino acid numbers. Full-

length Mdm2 and known motifs are also shown.

- 29 - survival and increase lifespan (van der Horst et al., 2004), a similar Sir2 function as observed in worms.

1.4.3. Ubiquitination and Deubiquitination of FOXO

1.4.3.1. Ubiquitination and Degradation of FOXO

The degradation of FOXO transcription factors is mediated by the ubiqutin- proteasome pathway. In the presence of insulin and other growth factors, FOXO proteins are relocalized from the nucleus to the cytoplasm and to be degraded via the ubiquitin– proteasome pathway. PKB activation is necessary for insulin promoted ubiquitin- mediated degradation of FOXO1 and FOXO3A via the proteasome pathway (Aoki et al.,

2004; Plas and Thompson, 2003).

As for insulin induced proteasomal degradation of FOXO1, insulin treatment decreases endogenous FOXO1 proteins in HepG2 cells and this decrease is suppressed

by proteasome inhibitors. FOXO1 is ubiquitinated in vivo and in vitro; insulin enhances its

ubiquitination in cells. In addition, the insulin signal to FOXO1 degradation is mediated by the PI3K pathway. FOXO1 mutates at the serine or threonine residues that are phosphorylated by PKB inhibit FOXO1 ubiquitination in vivo and in vitro. These data suggest that efficient ubiquitination of FOXO1 requires both phosphorylation and cytoplasmic retention in the cells (Matsuzaki et al., 2003).

Skp2 is an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex. It inhibits FOXO1 during tumor suppression through ubiquitin-mediated degradation but it only ubiquitinates and degrades FOXO1, not FOXO3A or FOXO4. The effect of Skp2 on

FOXO1 requires PKB-specific phosphorylation of FOXO1 at Ser-256. Decrease ubiquitination is seen in the mutant of FOXO1 in which all three PKB sites are replaced by alanine and FOXO1 is forced to remain in the cytoplasm, through a mutation in the

- 30 - NLS. In addition, when phosphorylated FOXO1 is confined to the nucleus by a mutation in the NES, FOXO1 ubiquitination is decreased (Huang et al., 2005). This study also confirmed that only cytoplasmic FOXO1 is successfully ubiquitinated by an E3 ubiquitin ligase and subsequently degraded.

IKKβ (I kappa B kinase β) also causes the proteasome-dependent degradation of

FOXO factors. IKKβ induces the phosphorylation of FOXO3A at Ser644, in the extreme

C-terminal portion of the molecule. This phosphorylation results in the ubiquitination and subsequent degradation of FOXO3A. Since IKKβ-induced tumorigenesis can be suppressed by overexpression of FOXO3A (Hu et al., 2004), the regulation of FOXO protein degradation by IKKβ may play an important role in tumorigenesis. However,

Ser644 is not conserved in other FOXO isoforms and is not present in worms and flies. It remains to be determined whether IKKβ phosphorylates and controls the other FOXO isoforms. It is possible that the degradation of FOXO isoforms is regulated by different protein kinases through other independent mechanisms.

Recently, it was showed that in the presence of oxidative stress FOXO4 becomes monoubiquitinated, resulting in its re-localization to the nucleus and increased transcriptional activity (van der Horst et al., 2006). This study demonstrated that besides

FOXO degradation, the ubiqutination of FOXO may also have other biological functions.

1.4.3.2. Deubiquitination of FOXO

USP7/HAUSP is a herpes virus-associated ubiquitin-specific protease.

Deubiquitination of FOXO4 requires the enzyme USP7/HAUSP, which interacts with and

deubiquitinates FOXO4 in response to oxidative stress. Oxidative stress-induced

ubiquitination and deubiquitination by USP7 do not influence the half-life of FOXO4

- 31 - protein. Moreover, USP7 does negatively affect FOXO transcriptional activity towards endogenous promoters (van der Horst et al., 2006).

2. Ubiquitin, Proteasome and MDM2 as an E3 Ligase

Ubiquitination is a reversible post-translational modification of cellular proteins, in

which ubiquitin is attached to the target proteins. Aberrations in the ubiquitination system

are implicated in the pathogenesis of many diseases, including certain malignancies, neurodegenerative disorders and pathologies of the inflammatory immune response.

Deubiqutination is a biological process in which one or more ubiqutin moieties are

removed from protein. Deubiquitination helps to maintain the pool of free ubiquitin.

2.1. Ubiquitin-Proteasome System

2.1.1. Ubiquitin and Ubiqutination

Ubiquitin (Ub) is a small protein composed of 76 amino acids. Ub is found only in

eukaryotic organisms and not in either eubacteria or archaebacteria. Among eukaryotes, ubiquitin is highly conserved. Ub is a heat-stable protein folded into a compact globular structure. Ub is found throughout the cell and can exist either in free form or as part of a complex with other proteins. In the latter case, Ub is conjugated to proteins through a covalent bond between at the C-terminal end of Ub and the side chains of lysine on the target proteins. A single Ub can be conjugated to the lysine of these proteins, or more commonly, Ub-chains can be attached. Uiquitination depends on ATP.

Ub is encoded by a family of genes whose translation products are fusion proteins. The Ub genes typically exist in two states: 1) The ubiquitin gene can be fused to a ribosomal gene giving rise to a translation product that is an Ub-ribosomal ; or 2) Ub genes can exist as a linear repeat, meaning that the translation product consists of a linear chain of Ub-molecules fused together (a polyubiquitin molecule). - 32 - After the fusion proteins are synthesized, another protein called Ub-C-term hydrolase cleaves the fusion proteins at the C-terminal end of Ub. This either liberates an individual

Ub and ribosomal protein or liberates a set of Ub monomers from the polyubiquitin.

Ubiquitination is a process in which ubiquitin is conjugated to the protein substrates. The attachment of a single ubiquitin polypeptide, monoubiquitin, to a substrate serves as an important regulatory modification (Hicke, 2001). Monoubiquitin acts as a sorting signal throughout the endocytic pathway and regulates diverse proteins

including histones, endocytic machinery and transcription factors. Monoubiquitination

targets proteins to the lysosome, either by directing endocytosis of cell-surface receptors

or by sorting newly synthesized hydrolases from the Golgi to their resident lysosomal

compartment (Lee and Tyers, 2001). A polyubiquitin chain is formed when ubiquitin is

attached to a lysine within ubiquitin itself and this process is repeated. Polyubiquitin

chains linked through different lysine residues are involved in distinct cellular functions.

For instance, the signal necessary for degradation of substrates by the proteasome is a

polyubiquitin chain attached through Lys-48, whereas chains linked through Lys-63 are

crucial to the role of ubiquitin in DNA damage repair (Pickart, 2001). Polyubiquitination

typically targets proteins for rapid proteasomal degradation. Polyubiquitination is also

critical in protein quality control, where it helps to eliminate improperly processed or mis-

folded proteins from the ER (endoplasmic reticulum), in a process called ER-associated

degradation (Lee and Tyers, 2001).

2.1.2. Deubiquitination

Both poly- and monoubiquitination can be reversed by DUBs (deubiquitinating enzymes) that specifically cleave the at the C terminus of ubiquitin.

DUBs also generate the pool of free ubiquitin both by liberating ubiquitin from precursor

- 33 - ubiquitin fusion proteins and by recycling ubiquitin from the branched polyubiquitin chains of degraded proteins (D'Andrea and Pellman, 1998; Wilkinson, 1997).

The DUBs are comprised of two groups of enzymes, the UCHs (ubiquitin C- terminal hydrolyases) and the USPs (ubiquitin-specific proteases; referred to as UBPs in yeast). The UCHs are small (20-30 kDa), closely related proteases that are generally involved in cleaving ubiquitin from small processed peptides. The USPs are more numerous, much larger (60–300 kDa), and are thought to have specific protein targets.

USPs can be identified by conserved sequences within the active site, but sequences outside of the catalytic domain are highly divergent, likely reflecting their role in mediating interactions with different protein targets (Hu et al., 2002; Wilkinson, 1997; Wilkinson et al., 1995).

There are 16 known UBPs in Saccharomyces cerevisiae, and 63 putative USP

genes in humans. Single and even multiple UBP deletions in yeast generally produce minimal phenotypic abnormalities, suggestive of functional redundancies among the yeast UBP family (Amerik et al., 2000). However, studies have shown that USPs can play specific roles in various biological processes in higher eukaryotes, suggesting a more specialized role as cellular regulators in multicellular organisms. Specific DUBs

have been shown to regulate eye development (Huang et al., 1995; Huang and Fischer-

Vize, 1996), cell growth in response to cytokines (Zhu et al., 1996), oncogenic transformation (Gray et al., 1995; Jensen et al., 1998; Papa and Hochstrasser, 1993), cell cycle regulation (Hu et al., 2002), chromatin structure (Robzyk et al., 2000), and transcriptional regulation (Holowaty et al., 2003; Mimnaugh et al., 1997; Moazed and

Johnson, 1996).

- 34 - 2.1.3. Ubiquitination Machinery and Proteasome Pathway

Ubiquitination of a protein substrate requires the concerted action of three classes of enzymes designated E1, E2, and E3. E1 (ubiquitin activating enzyme) initially activates ubiquitin in an ATP-dependent reaction through the formation of a thiol-ester bond between the carboxyl terminus of ubiquitin and the thiol group of a specific residue of E1. Ubiquitin is then transferred to a specific cysteine residue on one of several E2 (ubiquitin-conjugating enzymes, Ubcs). E2 enzymes in turn transfer the ubiquitin either directly to a substrate or to the final class of enzymes known as E3

(ubiquitin protein ligases). E3 enzymes catalyze the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and the amino group of lysine residues on the target protein. A substrate may then undergo multiply ubiquitinations through the attachment of additional ubiquitin molecules to specific lysine residues of ubiquitin itself.

In many cases the E1, E2, and E3 enzymes form large, multi-protein complexes. This increases the efficiency of the process by allowing the rapid thiol-ester transfer of ubiquitin molecules between proteins.

This type of protein degradation plays a role in many cellular processes, such as cell cycle regulation, antigen presentation, and the disposal of denatured, unfolded, or oxidized proteins. Most intracellular protein degradation is through the ubiquitin- proteasome pathway (Ciechanover and Schwartz, 2002; Hershko and Ciechanover,

1998).

In the ubiquitin-proteasome degradation pathway, the covalent attachment of multiple ubiquitin molecules to lysine residues of a target protein serves to signal its recognition and rapid degradation by the 26S proteasome. The proteasome is a large, multisubunit complex that exists in cells in two forms: a 20S and a 26S species. The

- 35 -

Figure 3 Ubiquitin-proteasome degradation pathway. E1 (ubiquitin activating enzyme) activates Ub in the presence of ATP. Activated Ub is then transferred to E2 (ubiquitin conjugating enzyme). E2 in turn transfers Ub to E3

(ubiquitin protein ligase). E3 binds ubiquitin to the substrate protein.

Ubquitinated proteins are degraded by the proteasome. Ub – ubiquitin.

- 36 - active protease sites sequestered in its central cavity, so that only proteins entering this chamber are degraded. The openings to this cavity permit only denatured proteins to enter, where they are progressively cleaved to small peptides. The addition of a 19S regulator to either or both ends of the 20S proteasome creates the 26S proteasome. The

19S regulator contains ATPases and other proteins and serves numerous functions, including the recognition of the substrate and its translocation to the catalytic core

(Voges et al., 1999).

2.2. MDM2 as an Ubiquitin E3 Ligase

2.2.1. General Information about MDM2

MDM2 is the product of the 'murine double minute 2' gene. The MDM2 gene was originally identified as one of three genes (mdm1, 2, 3) which were overexpressed by

more than 50-fold through amplification in a spontaneously transformed mouse BALB/c

cell line (3T3-DM). The mdm2 genes are located on small, acentromeric

extrachromosomal nuclear bodies, called double minutes, which are retained in cells

only if they provide a growth advantage. The gene product of mdm2 was later shown to

be responsible for cell transformation when MDM2 was overexpressed (Cahilly-Snyder

et al., 1987; Fakharzadeh et al., 1991). In 1992, Oliner cloned the human MDM2 gene

and mapped it to chromosome 12q13-14 (Oliner et al., 1992). Both the mdm2 gene and

its human counterpart, MDM2, consist of 12 exons that can generate many different

MDM2 proteins, as shown in Figure 4. There are two different promoters, the second of

which is responsive to p53. These promoters generate two proteins, the full-length p90

and a shorter p76 protein that initiates at an internal ATG (Olson et al., 1993; Perry et

al., 1993; Saucedo et al., 1999). p76 is missing part of the p53-binding domain and it can

act as a dominant negative inhibitor of p90 and activate p53. Alternative splicing of

- 37 - MDM2 to generate shorter proteins also occurs in many human and mouse tumors. More than 40 different alternatively spliced transcript variants have been isolated from both tumor and normal tissues. In humans, MDM2-a and MDM2-b are the major splice variants that delete exons 4–9 and 4–11, respectively. Neither product contains the p53-

binding motif. MDM2-b, also named MDM2-ALT1, interacts with full-length MDM2 and

sequesters it in the cytoplasm (Bartel et al., 2002).

2.2.2. Functions of MDM2

2.2.2.1. MDM2 and Cell Cycle

Overexpression of MDM2 has been shown to correlate with the cyclin-dependent kinase inhibitor p21. In breast cancer cells, overexpression of MDM2 correlates with lack

of p21 expression (Jiang et al., 1997). On the other hand, in squamous cell carcinoma,

overexpression of MDM2 is associated with high levels of p21 (Ng et al., 1999).

MDM2 reverses the growth inhibition at G1 imposed by p53 and RB (Chen et al.,

1996; Xiao et al., 1995). Overproduction of MDM2 can overcome the TGF-β-imposed growth inhibition via the RB– pathway (Sun et al., 1998).

Transgenic mice experiments showed that expression of a BLG (β- lactoglobulin)/mdm2 transgene (BLGmdm2) in the epithelial cells of the mouse mammary gland caused mammary epithelial cells to undergo multiple rounds of S phase without cell division, and resulted in polyploidy and tumor formation. The effect of MDM2 on S phase is independent of the p53 status (Lundgren et al., 1997).

2.2.2.2. MDM2 and Differentiation

One phenotype of tumor cells is the lack of terminal differentiation. MDM2 plays a very important role in epidermal differentiation (Dazard et al., 1997). MDM2 overexpression in the granular layer perturbs the differentiation program (Alkhalaf et al., - 38 - 1999). In rhabdomyosarcoma, forced expression of MDM2 inhibits MyoD function and consequently inhibits muscle differentiation (Fiddler et al., 1996). A MDM2-conditional mice experiment showed that MDM2 plays a very important role in bone organogenesis and homeostasis through inhibition of p53 function, which is a prerequisite for master osteoblast transcriptional regulator Runx2 activation, osteoblast differentiation, and proper skeletal formation (Lengner et al., 2006). NUMb is a cell fate determinant protein.

MDM2 associates with NUMb and influences cell differentiation and survival through translocation of NUMb to nucleus and degradation of NUMb (Juven-Gershon et al.,

1998).

However, microinjection of MDM2 mRNA in two-cell stage zebrafish embryos caused inhibition of cellular convergence during gastrulation. Clones derived from MDM2 microinjected blastomeres were significantly smaller than those derived from control microinjections. This indicates that MDM2 expression may be important during the differentiation of neural and muscular tissues of zebrafish (Thisse et al., 2000).

2.2.2.3. MDM2 and Ribosome Biogenesis

Ribosome biogenesis is the process of making ribosomes. It takes place both in the cell cytoplasm and in the nucleolus of eukaryotic cells. It involves the coordinated action of over 200 proteins in the synthesis and processing of the four rRNAs, as well as assembly of those rRNAs with the ribosomal proteins. Proper ribosome assembly is essential for the health of a cell.

Ribosome proteins L5, L11 and L23 exist in the same complex with MDM2 in response to ribosome stress, such as exposure to actinomycin D. They activate p53 by inhibiting MDM2-mediated p53 suppression (Dai et al., 2004; Lohrum et al., 2003;

Marechal et al., 1994; Zhang et al., 2003).

- 39 - 2.2.2.4. MDM2 and Transcription

MDM2 affects the gene transcription by inhibiting p53-mediated transactivation

(Momand et al., 1992). MDM2 uses multiple mechanisms to inactivate p53 and to inhibit its transcriptional activity. MDM2 targets p53 for ubiquitination and degradation by the proteosome, shuttles p53 out of the nucleus, prevents its interaction with transcriptional coactivators and recruits the known transcriptional corepressors, such as hCtBP2, to p53.

2.2.2.5. MDM2 and Protein Ubiquitination and Degradation

E3 ubiquitin ligases are a large family of proteins engaged in the regulation of the turnover and activity of many proteins. Together with ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, E3 ubiquitin ligases catalyze the ubiquitination of a variety of biologically significant protein substrates leading to their degradation through the 26S proteasome. Because they serve as the specific substrate-recognition element of the system, E3 ligases play an important role in the ubiquitin-mediated proteolytic cascade. There are approximately 1000 E3 ligases in the .

MDM2 possesses the activity of an E3 ubiquitin ligase. MDM2 was initially found to promote the proteasome–dependent degradation of p53 (Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997). It functions as an E3 ubiquitin ligase for p53 and for itself through its RING finger domain at the C-terminus (Fang et al., 2000; Honda et al.,

1997; Honda and Yasuda, 2000). It is now known that MDM2 also promotes the degradation of several other proteins in intact cells, such as: Numb (Yogosawa et al.,

2003), RB (Miwa et al., 2006; Uchida et al., 2005) and MDMX (Pan and Chen, 2003).

- 40 - 2.2.3. Regulation of MDM2 E3 Activity

2.2.3.1. Sumoylation of MDM2

2.2.3.1.1. SUMO and Sumoylation

SUMOs (small ubiquitin-related modifiers) constitute a family of highly conserved proteins found in all eukaryotes and are required for viability of most eukaryotic cells, including budding yeast, nematodes, fruit flies, and vertebrate cells in culture

(Apionishev et al., 2001; Epps and Tanda, 1998; Fraser et al., 2000; Hayashi et al.,

2002; Jones et al., 2002). In multicellular organisms, SUMO conjugation takes place in all tissues and at all developmental stages (Chen et al., 1998; Howe et al., 1998;

Joanisse et al., 1998; Kamitani et al., 1998; Kurepa et al., 2003; Lois et al., 2003;

Mannen et al., 1996; Shen et al., 1996). Since its discovery in 1996, SUMO has been found covalently attached to more than 50 proteins, including the ,

IκBα, c-Jun, HDACs and p53. Proteins that participate in transcription, DNA repair, nuclear transport, signal transduction and the cell cycle have been found to be sumoylated. In contrast to ubiquitination, however, sumoylation of a protein does not appear to target it for rapid degradation, but rather affect the ability of the modified protein to interact with cellular factors. Most SUMO-modified proteins that have been characterized in mammalian systems are involved in transcription and they are often repressed by SUMO conjugation. However, genetic studies in model organisms have pointed to a role for SUMO in chromosome dynamics and higher order chromatin structures, illustrating the diversity of SUMO function.

SUMO often has a positive effect on protein-protein interactions, and it promotes assembly of several multi-protein complexes. However, the effects of SUMO on interactions vary depending on the substrates. SUMO can also act by a completely

- 41 - different mechanism, including the prevention of ubiquitination of a protein by blocking lysine residue where Ub would normally be attached (Desterro et al., 1998; Hoege et al.,

2002; Lee et al., 2003; Lin et al., 2003a).

The linkage between SUMO and its substrates is an isopeptide bond between the C-terminal carboxyl group of SUMO and the ε-amino group of a lysine residue in the

substrate. A three-step enzyme pathway attaches SUMO to specific substrates, and

other enzymes cleave SUMO off its targets. The enzymes of the SUMO pathway,

although analogous to those of the Ub pathway, are specific for SUMO and have no role

in conjugating Ub or any of the other ubiquitin-like modifiers. The SUMO pathway begins

with a SUMO-activating enzyme (also called E1). E1 catalyses an ATP-dependent

activation of the SUMO C-terminus and then transfers activated SUMO to a SUMO-

conjugating enzyme (E2), also known as Ubc9. SUMO is then transferred from Ubc9 to

the substrate with the assistance of one of several SUMO-protein ligases (E3s). In

contrast to the ubiquitin-conjugating system, where E3 ligase is responsible for target

recognition, the recognition of SUMO targets is mediated by both E2 and E3 enzymes.

Many of the Lys residues where SUMO becomes attached are in the short consensus

sequence ψΩKXE/D, where ψ denotes a bulky aliphatic residue, Ω denotes a large

hydrophobic amino acid, generally isoleucine, leucine, or valine; K is the lysine residue

being modified; X is any residue; and E/D is a glutamic or . This motif is

bound directly by Ubc9. E3 ligases probably enhance specificity by interacting with other

features of the substrate. Although most known SUMO targets contain this sequence,

other conjugation sites are now beginning to be known, such as TKET in S. cerevisiae

PCNA (Hoege et al., 2002) and VKYC in Smad4 (Lee et al., 2003; Lin et al., 2003b).

Sumoylation is a reversible modification, and removal of SUMO is carried out by

enzymes that specifically cleave at the C-terminus of SUMO (Johnson, 2004). All known

- 42 - SUMO-cleaving enzymes belong to the family of ubiquitin like protease 1 (Ulp1) cysteine proteases and contain a 200 amino acid C-terminal core domain (the Ulp domain). The core domain has the SUMO cleaving activity and contains the catalytic triad Cys-His-Asn

(Mossessova and Lima, 2000). Mammals have seven members of the Ulp1 family:

SENP1-3 and SENP5-8. Only four of the SENP genes have been confirmed to encode

SUMO proteases, namely SENP1 (Bailey and O'Hare, 2002), SENP3 (SMT3IP1)

(Nishida et al., 2000), SENP6 (SUSP1) (Kim et al., 2000) and SENP2 (Axam,

SMT3IP2/Axam2, SuPr-1) (Best et al., 2002; Kadoya et al., 2002; Nishida et al., 2001).

2.2.3.1.2. Sumoylation and MDM2

SUMO-1 modification of MDM2 can differentially modulate the outcome of MDM2

E3 ligase activity in a manner that favors accmulation of p53. Upon Sumo-1 conjugation,

MDM2 is protected from self-ubiquitination and elicits greater ubiquitin-protein isopeptide ligase (E3) activity toward p53, thereby increasing its oncogenic potential. This switch in

modification status is stress-responsive, because UV irradiation leads to a decrease in the interaction of MDM2 with Ubc9 and a corresponding loss of MDM2 sumoylation

(Buschmann et al., 2001). Further studies showed that Ubc9 can associate with MDM2 only if amino acids 40-59 within the N-terminus of MDM2 are present. Furthermore, addition of a peptide corresponding to amino acids 40-59 of MDM2 efficiently inhibits

MDM2 sumoylation in vitro and in vivo (Buschmann et al., 2001)

2.2.3.2. Ubiqutination and Degradation of MDM2

As mentioned earlier, MDM2 mediates autoubiquitination as well as the

ubiquitination of other substrates. The balance between auto- and substrate-

ubiquitination of MDM2 is modulated physiologically by posttranslational modifications, including sumoylation and phosphorylation. After SUMO conjugation to MDM2, MDM2

- 43 - E3 ligase activity is shifted toward p53, while self-ubiquitination is minimized

(Buschmann et al., 2001).

P300 is an acetylase-possessing transcriptional co-activator that has been

shown to mediate transcription by numerous transcriptional activators. It binds to and

stabilizes MDM2. It stabilizes MDM2 by retaining it in a specific nuclear structure but

does not acetylate MDM2 in solution or in cells.

2.2.3.3. Phosphorylation of MDM2

The first demonstration of the complex nature of MDM2 was by

Henning who showed that the phosphorylation status of MDM2 was influenced by early gene expression of SV40. MDM2 is stabilized in the presence of SV40. Moreover,

hyperphosphorylated MDM2 participates in a trimeric complex with p53 and T-Ag (T-

antigen, the transforming protein of SV40), which is thought to activate oncogenic functions of MDM2 and enhance the transforming potential of T-Ag (Henning et al.,

1997).

Nearly 20% of the residues of MDM2 are either serine or threonine. MDM2 protein is phosphorylated at multiple sites in vivo. Two clusters of phosphorylation sites are located at the N-terminal (amino acids 1–193) and central amino acids 194–293 of murine Mdm2, respectively (Hay and Meek, 2000).

2.2.3.3.1. DNA-PK, ATM and MDM2

The PI3K family of enzymes generates lipid 'second messengers' that mediate signal transduction. It includes four classes of proteins. Class IV of PI3K includes mTOR

(mammalian target of rapamycin), DNA-PK (DNA activated protein kinase), ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases. MDM2 is

phosphorylated in vitro by both DNA-PK (Mayo et al., 1997) and ATM (Maya et al., 2001)

- 44 - but phosphorylation by ATR has not yet been reported. Of eight potential DNA-PK

targets in MDM2, only Ser17 has been shown to be phosphorylated by this enzyme in

vitro (Mayo et al., 1997). Although physiological phosphorylation of Ser17 has been confirmed, the phosphorylation site itself has been reported to have a significant impact

on the ability of MDM2 to regulate the response to p53. S17A mutant, mimics the form of MDM2, was significantly more effective in inhibiting p53- dependent transactivation in cultured cells than wild-type MDM2. Nuclear magnetic resonance studies also showed that MDM2 amino acids 16–24 can form a "flexible lid"

that folds over and stabilizes the MDM2 structure but competes only weakly with p53 for binding to this cleft. The S17D mutant peptide which mimics the consititutive phosphorylation form was found to have higher affinity for MDM2 than the wild-type peptide (McCoy et al., 2003).

ATM is able to phosphorylate MDM2 at Ser395 in vitro. In response to ionizing radiation and radiomimetic drugs, MDM2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation, which results in a decrease in its reactivity with the 2A10 monoclonal antibody. MDM2 S395D is impaired in promoting p53 degradation and it is markedly less able to promote p53 cytoplasmic export (Maya et al.,

2001).

2.2.3.3.2. PKB and MDM2

Mitogen-induced activation of PKB results in phosphorylation of MDM2 on Ser166

and Ser186. Phosphorylation on these sites is necessary for translocation of MDM2 from

the cytoplasm into the nucleus (Mayo and Donner, 2001). Moreover, phosphorylation of

MDM2 not only enhances its nuclear localization but its interaction with p300, and inhibits its interaction with p19ARF, resulting in increased p53 degradation (Zhou et al.,

2001). - 45 - PKB inhibits MDM2 self-ubiquitination via phosphorylation of MDM2 on Ser166

and Ser188. Stimulation of human embryonic kidney 293 cells with IGF-1 increased

MDM2 phosphorylation on Ser166 and Ser188 in a PI3K-dependent manner. Treatment of both human embryonic kidney 293 and COS-1 cells with PI3K inhibitor LY-294002 led to proteasome-mediated MDM2 degradation (Feng et al., 2004).

2.2.3.3.3. c-Abl and MDM2

c-Abl is an non-receptor tyrosine kinase that can shuttle between the cytoplasmic and nuclear compartments. In response to stress, such as DNA damage, c-Abl promotes cell growth arrest and apoptosis. The apoptotic activity of c-Abl is mediated partly via (Agami et al., 1999; Gong et al., 1999) and to a lesser extent through a p53- dependent pathway (Yuan et al., 1997). One mechanism for the protection of p53 by c-

Abl is that c-Abl can neutralize the ability of MDM2 to ubiquitinate p53 and degrade it

(Sionov et al., 1999). C-Abl directly interacts with MDM2 at multiple sites in the nucleus, enhances its accumulation in a p53-independent manner. c-Abl phosphorylates MDM2 at Tyr394. Substitution of Tyr394 by Phe394 enhances the ability of MDM2 to promote p53 degradation and to inhibit the transcriptional and apoptotic activities of p53

(Goldberg et al., 2002).

2.2.3.3.4. CK2 and MDM2

Protein kinase CK2 is a ubiquitous Ser/Thr protein kinase required for cell cycle progression and cell viability. Serine residue at position 269 of MDM2 was established as the most important CK2 phosphorylation site by analyses with deletion mutants of

MDM2 and a peptide library. Phosphorylation of MDM2 by CK2 is stimulated in the

presence of the C-terminal sequences of p53, but binding studies revealed that the

- 46 - P1 P2 ATG ATG

1 27 28 52 53 97 97 114 114 133 134 166166 220 221 272 273 298 299 491 I II III IV V VI VII VIII IX X XI XII

1 26 108 178 192 230 274 289 331 433 467 471 491

MDM2 P P P P P P P P NoLS NLS NESAcidic Zn- Ring-finger domain finger S17 S166 S186 S188 S269 S395 DNA-PK PKB/AKT CK2 ATM T216 Y394 CyclinA/CDK2 c-Abl

1 27 223 491 MDM2-a 1 27 301 491 MDM2-b 1 52 223 491 MDM2-c 1 27 389 491 MDM2-d 1 75 483 491 MDM2-e 26 108 211 361 P53 P53 1 150 210 224 p73 ARF 1 134 221 294 Numb SP1,RB 102 222 341 491 P300 AR 1 222 284 374 E2F, TFIIE L5,L11 167 304 MTBP 50 166 202 304 340 488 DNA polymerase ε PML PML 50 384 PCAF

Figure 4 Structure of MDM2 gene and protein - 47 - Figure 4 Structure and regulation of MDM2 gene and protein. MDM2 gene consists of 12 exons. Two promoters are shown by arrows. Full-length MDM2 p90 is translated from the first start codon ATG in exon 3 and the short form, p76, is translated from the second ATG in exon 4. Phosphorylation sites are indicated by the letter P within an ellipse. Their locations relative to the functional domains of

MDM2 are shown. Protein kinases, where known, are indicated in boxes with the target residue(s) shown above. Five major alternative splice variants MDM2-a,

MDM2-b, MDM2-c, MDM2-d, and MDM2-e are shown. Full-length Mdm2 and known motifs are also represented. NLS-nuclear localization signal; NES-nuclear export signal; Zn-finger- domain; NoLS-nucleolar localization signal;

RING-finger-ring finger domain. The numbers above the drawings denote amino acid numbers and roman numerals denote the exon numbers.

- 48 - biological function of CK2 phosphorylation still needs to be established by further studies because phosphorylation of MDM2 at Ser269 does not have any influence on the binding of p53 to MDM2 (Gotz et al., 1999).

3. Functional interaction among FOXO, p53, and MDM2

3.1. Interaction between MDM2 and p53

P53 is a transcription factor that regulates the cell cycle and functions as a tumor suppressor. p53 has been described as "the guardian of the genome" or the "master watchman", referring to its role in conserving stability by preventing genome mutations.

In the absence of genetic damage, p53 is a very unstable protein with a half-life ranging from 5-30 min and transcriptional activity is inert (Yuan et al., 1996). In the presence of stress, such as DNA damage, hypoxia, telomere shortening, and oncogenic activation, p53 becomes stable and activated by blocking its degradation (Caspari, 2000; Meek,

1994; Sakaguchi et al., 1998; Siliciano et al., 1997). p53 can kill cells via dual

transcription-dependent and -independent functions in the nucleus and mitochondria

(Mihara et al., 2003; Vousden and Lu, 2002).

The p53-MDM2 system forms a feedback loop, in which p53 upregulates MDM2

by activating MDM2 transcription (Barak et al., 1993). Experiments with knock-out mice revealed that deletion of the mdm2 gene results in embryonic lethality, which can be rescued by deletion of the p53 gene (Jones et al., 1995; Montes de Oca Luna et al.,

1995). Inhibition of cell growth and marked cell death is often seen in the absence of p53 regulation by MDM2, further emphasizing the importance of the p53–MDM2

autoregulatory loop in controling of cell growth and death.

MDM2 negatively regulates p53 in several ways:

- 49 - 1) MDM2 binds to p53 and this interaction is conformation based. Site-directed experiments have demonstrated the importance of p53 residues Leu14, Phe19, Leu22, and Trp23 (Lin et al., 1994). A minimal MDM2-binding site on p53 residues 18-23 was mapped with p53-derived peptides (Picksley et al., 1994). On binding to the p53 transactivation domain, MDM2 inhibits its transcriptional activity. Crystallographic data showed that the amino terminal domain of MDM2 forms a deep hydrophobic cleft into

which the transactivation domain of p53 binds, thereby concealing itself from interacting with the transcriptional machinery (Kussie et al., 1996).

2) MDM2 functions as an E3 ubiqutin ligase for p53 (Honda and Yasuda, 1999;

Lohrum et al., 2000; Tao and Levine, 1999; Weber et al., 1999). The level of MDM2 is very important for the ubiquitination level of p53. In vitro studies showed that low levels of MDM2 activity induce monoubiquitination, whereas high levels promote polyubiquitination and nuclear degradation of p53 (Li et al., 2003a).

3) MDM2 promotes the export of p53 from the nucleus. p53 shuttles between nucleus and cytoplasm in the cells in response to stress. MDM2 contains an NES, but the study showed that the MDM2 RING finger domain, not the MDM2 NES, is necessary

for the efficient export of p53 to the cytoplasm (Boyd et al., 2000; Geyer et al., 2000).

Another study also showed that low MDM2 levels induce cytoplasmic translocation of

p53 (Li et al., 2003a), whereas MDM2-mediated p53 monoubiquitylation promotes its

mitochondrial translocation (Marchenko et al., 2007).

3.2. Interaction between p53 and FOXO

p53 and FOXO factors share similar characteristics. Both are involved in stress

response and can be post-translationally modified by phosphorylation and acetylation.

They also have some common downstream targets, such as: Fas ligand (Greer and

- 50 - Brunet, 2005), GADD45 (Greer and Brunet, 2005), PA26 (Greer and Brunet, 2005), p21

(Seoane et al., 2004) and PUMA (You et al., 2006a) (Figure 5).

Recently it was shown that p53 and FOXO3A interact with each other. In response to oxidative stress, p53 binds to FOXO3A. In vivo, these two transcription factors exhibit “crosstalk”. In response to DNA damage, p53 activation leads to FOXO3A phosphorylation and subcellular localization change, which results in inhibition of

FOXO3A transcription activity. PKB is dispensable for p53-dependent suppression of

FOXO3A. By contrast, SGK1 was significantly induced in a p53-dependent manner after

DNA damage, and this induction is through extracellular signal-regulated kinase 1/2- mediated posttranslational regulation. Furthermore, inhibition of SGK1 expression by a small interfering RNA knockdown significantly decreased FOXO3A phosphorylation in response to DNA damage.

Nuclear activated FOXO3A can impair p53 transcriptional activity. However, activation of FOXO3A either by serum starvation or by expressing a constitutively active form of FOXO3A can induce p53-dependent apoptosis, even in cells bearing a transcriptionally inactive form of p53 (You et al., 2006b).

- 51 -

DNA damage DNA damage

Oncogen Oxidative Oncogen Hypoxia activation Stress activation

FOXOs p53

P21 Fas Ligand P21 Fas Ligand P27 Bim PUMA 14-3-3δ PTEN PUMA GADD45 TRAIL GADD45 BAX Noxa

Cell cycle Arrest Apoptosis Cell cycle Arrest Apoptosis

Figure 5 Stress-induced FOXO and p53 pathway.

- 52 -

HYPOTHESIS & OBJECTIVES

FOXO factors are known to be ubiquitinated, but so far no general E3 ubiquitin ligases capable of ubiquitination of all FOXO factors have been identified. Skp2 is reported to promote the ubiqutination and degradation of FOXO1, but this effect is limited to FOXO1. A genetics study in C.elegans showed that skp expression is actually needed for FOXO transcriptional activity.

Since MDM2 is known to be an E3 ubiquitin ligase for p53 and both p53 and

FOXO factors are important regulators in stress responses, aging and tumorigensis, we, therefore, hypothesize that MDM2 interacts with and promotes the ubiquitination and degradation of FOXO factors.

To substantiate the hypothesis, my thesis studies are to achieve the following objectives:

1. Determine whether FOXO factors and MDM2 interact;

2. Determine whether MDM2 regulates the transcriptional activity of FOXO

factors;

3. Determine whether MDM2 decreases the stability of FOXO factors;

4. Determine whether MDM2 changes the biological function of FOXO;

5. Identify the signals that regulate effect of MDM2 on FOXO and investigate

the functional relationship among p53, FOXO and MDM2.

- 53 -

MATERIALS AND METHODS

Chemicals, Antibodies and Cell Lines:

Cycloheximide and nicotinamide were purchased from Sigma and MG132 from

Calbiochem. Antibodies against FOXO1 (H-128, Santa Cruz Biotechnology), FOXO3A

(H-144, Santa Cruz Biotechnology), MDM2 (SMP14, Santa Cruz Biotechnology; 2A10,

Calbiochem), c- (A-14, Santa Cruz Biotechnology), Flag (M2-A-2220 and F-7452,

Sigma), HA (MMS-101P and PRB-101P, Covance), MnSOD (Upstate), TRAIL (BD

Pharmingen), p27 (N-20, Santa Cruz), acetyl-K (Upstate), α-Tubulin (Sigma), and β-actin

(AC-74, Sigma) were purchased from commercial sources.

H1299/V138 cells were cultured as described (Pochampally et al., 1999). HEK

293T, NIH 3T3, DU145, JCA1, PC3, HeLa, MCF-7, Saos-2, H1299, p53 null MEFs

(mouse embryonic fibrblasts )and p53 and MDM2 double null MEFs (Huang et al., 2005;

Peng et al., 2001) were maintained in DMEM (Dulbecco's modified Eagle's medium) with

10% FBS (fetal bovine serum). LNCaP cells were cultured in RPMI 1640 with 10% FBS

at 37°C. H1299 cells stably expressing human MDM2 were generated by cotransfecting

MDM2 with pcDNA3 and selecting in the presence of 750 μg/ml G418.

Plasmids:

pcDNA3-Flag-FOXO1, pcDNA3-Flag-FOXO1 (AAA), HA-FOXO3A, HA-FOXO3A

(AAA) (Li et al., 2003b), HA-SIRT1 (Yang et al., 2005), HA-SIRT1(734R) (Yang et al.,

2007), pcMDM2 and different MDM2 mutants were described previously (Armoni et al.,

- 54 - 2006; Chen et al., 1995; Chen et al., 1993; Freedman and Levine, 1999). To construct pcDNA3-HA-FOXO1 (1-150), FOXO1 cDNA fragment coding the first 150 amino acids were amplified by PCR with primers 5'-CGG GGG TCA CCG GAT CCA TGG CCG AGG

C-3' and 5' –GCG GCG GGA CGA TCT AGA CTA GCG CGG CTG C-3', which

generated BamHI and XbaI sites at 5' and 3' ends of the DNA fragment, respectively.

The amplified FOXO1 (1-150) fragment was cloned into the BamHI and XbaI sites of

pcDNA3.1 HA vector (Invitrogen). pcDNA3-HA-FOXO1 (1-270) and pcDNA3-HA-FOXO1

(256-655) were constructed similarly by generating a BamHI site at the 5' end and a XbaI site at the 3' end of the corresponding FOXO1 cDNA fragments. The upstream primer of

FOXO1 (1-270) was same as the FOXO1 (1-150), and the downstream primer was 5'-

CTT GGC TCT AGA AGC TCG GCT TCG GCT CTT AG -3'. The upstream primer of

FOXO1 (256-655) was 5'-GGA GAA GAG CTG GAT CCA TGG ACA ACA AC-3' and the

downstream primers was 5'-CGG GCC CTC TAG ATC AGC CTG ACA CC -3'. MDM2

siRNAs were subcloned into pSilencer-Neo (Ambion). The corresponding

oligonucleotides for generating the MDM2 siRNA were 5'-GAT CCG CAG GTG TCA

CCT TGA AGG TTT CAA GAG AAC CTT CAA GGT GAC ACC TGT TTT TTG GAA A-

3'and 5'-GAT CCG TGG TTG CAT TGT CCA TGG CTT CAA GAG AGC CAT GGA CAA

TGC AAC CAT TTT TTG GAA A-3'. The oligonucleotides for GFP siRNA were from

Ambion.

Transfections and Immunological Assays:

For co-precipitation analysis, 106 cells were plated in 100 mm dishes in a

medium containing 10% fetal bovine serum. One day after plating, cells were transfected

with the indicated plasmids by Lipofectamine Plus following the protocol from Invitrogen.

Cellular extracts were prepared in a buffer containing 20 mM Tris-HCl (pH 7.5), 0.5% NP-

40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 - 55 - mM benzamidine and 1 mM phenylmethylsulfonyl fluoride. After pre-clearance by incubating with protein A-agarose for 1 hour (h) followed by brief centrifugation, the extracts were incubated sequentially with 1-3 µg antibody and protein G-agarose beads

for 4 h at 4°C. After four times washes with the lysis buffer, the immunoprecipitates were

eluted from the beads by boiling in sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) loading buffer.

For immunoblotting, cellular extracts or immunoprecipitates were separated on

SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, probed with the cognate antibody and visualized with enhanced chemiluminescence.

For immunofluorescence analyses, H1299 cells or MEFs transfected cells on cover slips were cultured in DMEM for 16 h, washed once with PBS (phosphate buffered saline) and fixed in 4% paraformaldehyde for 10 min at 37ºC. The cells were permeabilized in buffer containing 1% Triton X-100 and 1% BSA at room temperature for

30 min and incubated for another 1 h in PBS containing 0.2% NP-40, 1% bovine serum albumin and the primary antibody. After washing three times in PBS, the cells were incubated for 45 min with goat anti-mouse IgG conjugated with Alexa Fluor 594 (red) or fluorescein isothiocyanate (FITC; green) conjugated anti-rabbit IgG (Molecular Probes), respectively and washed three times with PBS. The slides were dried and mounted with

Vectashield mounting medium with DAPI (4', 6'-diamidino-2-phenylindole). DAPI staining was performed to visualize the nucleus. Regular fluorescent microscopic images were obtained with a Nikon Diaphot microscope using a Photometrix PXL cooled CCD camera. The microscope was equipped with the appropriate filters for three-color imaging and a motorized stage for obtaining z-series images. Digital image files were processed and deconvolved using the Oncor Image software (Oncor Inc.). High- resolution images of the deconvolved and 3-D reconstructed image z-series stacks were

- 56 - processed for presentation with Adobe Photoshop. For confocal analysis, samples were viewed with a Leica DMI6000 inverted microscope, TCS SP5 confocal scanner, and a

100X/1.40NA Plan Apochromat oil immersion objective (Leica Microsystems, Germany).

405 Diode and HeNe 594 laser lines were applied to excite the samples and tunable filters were used to minimize spectral overlap between fluorochromes. DIC imaging was performed using an argon laser line. Scale bars were created with the LAS AF software version 1.6.0 build 1016 (Leica Microsystems, Germany).

Ni-NTA Pull-down Assay:

H1299 cells or MEFs were plated in 100 mm dishes and transfected with 4 µg

His6-ubiquitin (His-Ub) plasmid, 4 µg FOXO vectors and 4 µg MDM2 vectors using

Lipofectamine Plus; 24 h post transfection, cells were harvested and separated into two

aliquots. One aliquot (10%) was subjected to immunoblotting analysis to detect the expression of transfected proteins. The other aliquot of cells (90%) was used to purify the proteins of interested using Ni2+-nitrilotriacetic acid (NTA) beads. Cell pellets were lysed in a buffer containing 0.01 M Tris-Cl (pH 8.0), 6 M guanidinium-HCl, 0.1 M sodium phsophate, 5 mM imidazole, 10 mM β-mercaptoethanol and incubated with Ni2+-NTA

beads (Qiagen) for overnight at room temperature. The beads were washed sequentially

with the lysis buffer, a buffer containing 0.01 M Tris-Cl (pH 8.0), 8 M urea, 0.1 M sodium

phsophate, 10 mM β-mercaptoethanol, and a buffer containing 0.01 M Tris-Cl (pH 6.3), 8

M urea, 0.1 M sodium phsophate, 10 mM β-mercaptoethanol. Proteins bound to the

beads were eluted with a buffer containing 0.15 M Tris-Cl (pH 6.7), 5% SDS, 200 mM

imidazole, 30% glycerol, 0.72 M β-mercaptoethanol and were subjected to

immunoblotting analysis for the presence of Ub-conjugated FOXO proteins.

- 57 - In vitro Transcription Coupled Translations and GST (Glutathione S-transferase)

Pull-down assays:

FOXO1 protein was produced with pcDNA3-Flag-FOXO1 as a template using T7 polymerase-based in vitro transcription coupled translations (Promega, Madison, Wis.).

GST-MDM2 plasmids were transformed into BL21 and cultured at 37°C until the optical

density at 600 nm reached 0.6. Then, 0.2 mM of isopropylthiogalactopyranoside (IPTG)

was added and the incubation continued for another 5 h at 30°C. Bacterial cultures were lysed by sonication in a buffer containing 50 mM Tris (pH 8.0), 10 mM NaCl, 1 mM

EDTA, 6 mM MgCl2, 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride. GST pull down analyses were performed using the MagneGST pull-down system (Promega

Madison, Wis) following the vendor’s protocols.

In Vitro Ubiquitination Assays:

Full length GST-MDM2 and GST-MDM2-NT (1-150) were expressed in E. coli and bound to glutathione agarose beads. The substrate FOXO1 was produced by in vitro transcription coupled translation in rabbit reticulocyte lysate using the TNT system

(Promega) in the presence of 35S- . 4 µg GST-fusion proteins and 8 µl FOXO1 in vitro translation product were incubated to allow the enzyme-substrate interaction to occur. After three times washes with PBS containing 0.2% NP-40, the bead-bound enzyme-substrate complex was incubated at 37°C for 1 h with 250 ng GST-Ubc5Hb

(Boston Biochem), 250 ng purified rabbit E1 (Boston Biochem), 2 µg His6-Ub (Boston

Biochem) in 20 µl reaction buffer containing 50 mM Tris (pH 7.5), 2.5 mM MgCl2, 15 mM

KCl, 1 mM dithiothreitol, 0.01% Triton X-100, 1% glycerol and 8 mM ATP. The reactions

were terminated by boiling in SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis. Gel was dried and the Ub-conjugated FOXO1 proteins were detected by autoradiography. - 58 - Apoptotic Analysis and Flow Cytometry:

The determination of the survival and apoptotic index of GFP (green fluorescent protein)-transfected cells has been described (Li et al., 2001). In brief, transfected cells

were washed with PBS, fixed in 4% formaldehyde and stained with DAPI.

Representative micrographs were captured by a charge-coupled device camera with a

Smart Capture Program (Vysis, Downers Grove, Ill.) attached to a Leitz Orthoplan 2 fluorescence microscope. The viability of transfected cells in each well was determined

by counting the total number of green cells in each well. The apoptotic index of GFP- positive cells was determined by scoring 300 GFP-positive cells for chromatin condensation and apoptotic body formation.

To assay the apoptosis index induced by FOXO1, H1299 cells in 100 mm dishes were transfected with GFP and FOXO1 with or without MDM2. Transfected cells were collected in PBS containing 2.5 mM EDTA, washed twice with cold PBS, re-suspended

in 1× binding buffer containing 0.01 M Hepes (pH7.4), 0.14 M NaCl, 2.5 mM CaCl2 at a

concentration of 1 × 106 cells/ml and stained with Annexin-V APC and 7-AAD. Cell sorting and flow cytometry analysis were performed on a FAC Scan (Becton Dickinson,

Mountain View, Calif.).

- 59 -

RESULTS

FOXO proteins are ubiquitinated and their expression level is regulated by

proteasome mediated degradation (Aoki et al., 2004; Plas and Thompson, 2003), but a

general ubiquitin E3 ligase for FOXO proteins remains to be identified.

Skp2 was reported to mediate the ubiquitination and degradation of FOXO1 but

such activity appears to be restricted to FOXO1 (Huang et al., 2005). So far, no

mutational analysis and in vitro enzyme assays have been performed to show the direct

involvement of Skp2 E3 activity in FOXO ubiquitination. It remains to be determined

whether Skp2 promotes FOXO1 ubiquitination directly, through its intrinsic E3 activity, or

indirectly through another E3. The present study showed that MDM2 is the E3 ubiquitin

ligase for FOXO factors. MDM2 interacts with FOXO, promotes the degradation and

affects downstream targets and the biological function of FOXO.

1. MDM2 promotes the degradation of FOXO factors.

In an effort to investigate the interaction between p53 and FOXO signaling pathways, an inverse correlation was noticed between the expression of FOXO3A and

the p53 target gene MDM2 using a panel of human cancer cell lines including

osteosarcoma (Saos2), prostate (LNCaP, PC3, DU145, and JCA1), breast (MCF-7),

cervical (Hela) and lung (H1299, H1299/V138) cancer cells (Figure 6). To determine whether the inverse correlation of these two proteins among cancer cells reflects a negative effect of MDM2 on FOXO protein, the level of FOXO3A and FOXO1 protein expression in p53 null and p53/MDM2 double null MEFs was measured by - 60 - immunoblotting. As shown in Figure 7, the knockout of MDM2 in MEFs resulted in an increased expression of endogenous FOXO1 and FOXO3A proteins. Consistent with the data from MEFs, the stable expression of MDM2 in H1299 cells reduced (Figure 8) and the knockdown of endogenous MDM2 increased (Figure 9) the expression of endogenous FOXO3A. Unlike MEFs, H1299/V138 cells express relatively low levels of endogenous FOXO1, which is difficult to detect. Thus, we tested the effect of MDM2 silencing on the expression of ectopic FOXO1. As shown in Figure 10, the knockdown of

MDM2 by shRNA increased the level of ectopic Flag-FOXO1 expression.

Because MDM2 is an E3 ubiquitin ligase that promotes proteasome mediated degradation of p53 (Honda et al., 1997) and other (Lin et al., 2002a) proteins, it is likely that MDM2 decreases the expression of FOXO factors in a proteasome-dependent manner. In transient transfection studies, co-expression of MDM2 reduced the expression level of Flag-FOXO1 in DU145 prostate cancer cells, a process that was prevented by treatment with two different concentrations of the proteasome inhibitor

MG132 (Figure 11).

To test whether the decreased FOXO protein expression by MDM2 is due to protein degradation, the half-life of Flag-FOXO1 was measured in H1299 cells cotransfected with control vector, full length MDM2 or an MDM2 (1-361) mutant which lacks the C-terminal E3 ligase domain. As shown in Figure 12, the half life of FOXO1 was decreased by full length MDM2 from 6 h to 3 h, as compared to the control vector.

When FOXO1 was cotransfection of MDM2 (1-361), MDM2 (1-361) significantly extended the half-life of Flag FOXO1; the exact meaning of the latter is unclear. Overall, the data raise the possibility that MDM2 may function as an E3 ubiqitin ligase for mammalian FOXO factors.

- 61 -

MW

(kD) LNCaP Saos2 JCA1 H1299/V-138 H1299 PC3 MCF7 DU145 Hela

105 MDM2

78 FOXO3A

Figure 6 Inverse correlation of MDM2 and FOXO3A protein expression in different cancer cell lines. DU145, JCA, H1299-V138, HeLa, LNCaP,

MCF7, PC3, Saos-2, H1299 cells were plated into 100 mm dishes. MDM2 and FOXO3A proteins were detected by Western blotting with anti-MDM2

(SMP-14 ) and anti-FOXO3A (H-144) antibodies, respectively.

- 62 - P53-/- MDM2-/- P53-/-

150 IB:MDM2 100 MDM2 75

50

IB:FOXO3A 150

100 FOXO3A 75

50

IB:FOXO1 FOXO1

β-actin IB:β-actin

Figure 7 Inverse correlation of MDM2 and FOXO protein expression in MEFs. p53-/- MEFs and p53-/-, MDM2-/- double null MEFs were plated in 100 mm dishes.

The cells were harvested the next day and cell lysates were subjected to immunoblotting analyses with anti-MDM2 (SMP-14 ) antibody, anti-FOXO1 antibody, and anti-FOXO3A (H-144) antibodies, respectively. The β-actin level showed the equal loading. - 63 -

H1299 Stable Clones Control MDM2-1 MDM2-2

IB:MDM2 MDM2

IB:FOXO3A FOXO3A

IB:β-actin β-actin

Figure 8 Stably expressed MDM2 decreases the level of endogenous

FOXO3A expression. H1299 cells stably expressing MDM2 (MDM2-1, MDM2-

2) or empty vector (Control) were established and cell extracts were subjected to

Western blot analyses with the indicated antibodies.

- 64 -

siRNA GFP MDM2-2 MDM2-1

IB:MDM2 MDM2

IB:FOXO3A FOXO3A

β IB:β -actin -actin

Figure 9 Knockdown of endogenous MDM2 by siRNA causes an increase in endogenous FOXO3A protein. H1299 cells were transfected with control GFP-siRNAs or siRNAs (MDM2-siRNA1, MDM2-siRNA2) for

MDM2, and cell extracts were subjected to Western blot analyses.

- 65 -

- - + + Flag-FOXO1 + - + - Control-shRNA - + - + MDM2-shRNA

IB:MDM2 MDM2

IB:M2 Flag-FOXO1

β-actin IB: β-actin

Figure 10 Knockdown of endogenous MDM2 by siRNA increases the level of ectopic FOXO1 protein. A Flag-tagged FOXO1 expression vector was cotransfected transiently with MDM2-siRNA or control siRNA

(GFP-siRNA) into H1299-V138 cells, and whole cell extracts were prepared and subjected to Western blot analyses.

- 66 -

Flag-FOXO1 - + + + + + + MDM2 - - + - + - + MG132 (μM) 0 0 0 5 5 10 10

IB:M2 FOXO1

IB:MDM2 MDM2

IB:β-Actin β-Actin

1.5

1

0.5

FOXO1 Protein Density Protein FOXO1 0 1234567

Figure 11 MDM2 overexpression causes a decrease in Flag-FOXO1 protein, which is blocked by MG132. DU145 cells were transfected with Flag-FOXO1 and pcMDM2. 24 h posttransfections, cells were incubated with or without different concentrations of MG132 for 6 h. Cell lysates were separated in a 8% SDS-PAGE.

FOXO1 and MDM2 were detected by Western blotting with anti-Flag (M2) and anti-

MDM2 (SMP-14) antibodies, respectively. Equal amounts of protein were loaded.

- 67 -

Flag-FOXO1 - + + + + + + + + + + + + + + + MDM2 ------+ + + + + - - - - - MDM2(1-361) ------+ + + + + CHX treatment (h) - 0 3 6 12 24 0 3 6 12 24 0 3 6 12 24

Flag- FOXO1

β-actin

CTL 120 MDM2 MDM2( 1- 361) 100 –actin (%)

β 80

60

FOXO1/ FOXO1/ 40

20 (hrs) 0 0 3 6 12 24

Figure 12 MDM2 Overexpression results in a decrease in half-life of the

FOXO1 protein. H1299 cells were transfected with the plasmids as indicated. 24 h

after transfection, cells were treated with CHX for different times. The cell lysates

were separated by SDS−8% PAGE. Exogenous FOXO1 was detected by Western

blotting with anti-Flag M2 antibodies. Equal amounts of protein were loaded on each

sample.

- 68 - 2. MDM2 interacts with FOXO factors in vivo and in vitro.

Ubiquitin E3 ligases are known to make direct contact with their substrates.

Therefore, it was investigated whether or not MDM2 and FOXO factors form a complex

in cells. Deconvolution imaging analysis detected ectopic MDM2 and FOXO1 proteins in

both cytoplasmic and nuclear compartments of p53/MDM2 double null MEFs, but

colocalization was detected predominantly in the nucleus (Figure 13). Similarly, confocal

imaging gave the same result for endogenous MDM2 and FOXO3A in H1299 cells

(Figure 14).

To determine whether MDM2 and FOXO1 interact in mammalian cells, they were

ectopically expressed in H1299 cells and reciprocal coimmunoprecipitations performed.

MDM2 and FOXO1 were co-precipitated in cells expressing both proteins. In cells that

express either MDM2 or FOXO1, little or no coprecipitations were observed, suggesting

that co-precipitations were not due to cross reactivity of the antibodies (Figure 15).

Similar analysis showed that MDM2 and GFP-FOXO3A were specifically co-precipitated,

but not MDM2 and GFP (Figure 16).

To determine whether endogenous MDM2 and FOXO1 interacted in mammalian

cells, H1299/V138 cells expressing a temperature-sensitive p53 mutant (Pochampally et

al., 1999) were shifted to permissive temperature for 16 h to induce MDM2 expression

and cellular extracts were subjected to coimmunoprecipitation analysis. As shown in

Figure 17, FOXO1 was co-precipitated by the anti-MDM2 antibody, but not by the control

antibody, showing that the co-precipitations were not due to antibody cross reactivity.

To determine whether endogenous MDM2 and FOXO3A form a complex,

extracts of HEK293T cells were treated with DMSO or MG132 and co-

immunoprecipitations with an anti-MDM2 antibody. An anti-HA antibody was

- 69 -

Merged

20 microns

MDM2 Flag-FOXO1

Figure 13 Exogenous MDM2 and Flag-FOXO1 are colocalized in the nucleus of MEF cells. p53-/-, MDM2-/- MEF in a medium containing 0.5% FBS were transfected with 1 μg pcMDM2 and 1 μg Flag-FOXO1. Cells were stained with DAPI and incubated with anti-MDM2 (SMP14) and anti-FOXO1 (H-128) antibodies.

Immunoreactivity was detected with IgG conjugated to Alexa Fluor 594 (red for

MDM2) or FITC (green for FOXO1). Colocalization was determined by high- resolution imaging with deconvolution microscopy.

- 70 -

Merged

20 microns

DAPI MDM2 FOXO3A

Figure 14 Endogenous MDM2 and FOXO3A are colocalized in the nucleus of

H1299 cells. H1299 cells in a medium containing 1% FBS were stained with DAPI and incubated with anti-MDM2 (SMP14) and anti-FOXO3A (H-144) antibodies.

Immunoreactivity was detected with IgG conjugated to Alexa Fluor 594 (red for

MDM2) or FITC (green for FOXO3A). Colocalization was visualized by high- resolution imaging with confocal microscopy.

- 71 -

+ - + HA-FOXO1 MW (kD) - + + MDM2

IP: HA 109 IB: MDM2 MDM2 78

60

109 IP:MDM2 IB:HA 78 FOXO1

60

IB: MDM2 MDM2

IB: HA FOXO1 78 60 47 IB: β-actin β-actin

Figure 15 Interaction of ectopic FOXO1 and MDM2 in H1299 cells.

H1299 cells were cotransfected with HA-tagged FOXO1 and pcMDM2. 24 h posttransfection, cell extracts were immunoprecipitated with either anti-HA or anti-MDM2 antibodies followed by immunoblotting with anti-MDM2 or anti-HA antibody.

- 72 -

- + GFP-FOXO3A + - GFP MW + + MDM2 (kD) FOXO3A IP: MDM2 78 IB: GFP 60

47 36 24 MDM2 IP: GFP IB: MDM2

IB:MDM2 MDM2

FOXO3A IB:GFP

GFP IB:GFP

Figure 16 Interaction of ectopic FOXO3A and MDM2 in HEK293T cells.

HEK293T cells were cotransfected with pcMDM2 and either GFP or GFP-

FOXO3A. 24 h posttransfection, cell extract were immunoprecipitated with either an anti-MDM2 or an anti-GFP antibody followed by immunoblotting with an anti-

GFP or an anti-MDM2 antibody.

- 73 -

- + +MG132 M M HA IP MW (kD)

IB: FOXO1 75 FOXO1

50

IB: MDM2 MDM2 75

Heavy Chain 50

Figure 17 Interaction of endogenous MDM2 and FOXO1 in H1299/V138 cells. Cells were shifted from 39°C and cultured in 33°C for overnight and were treated with or without MG132 for 6 h. Cell extracts were immunoprecipitated with an anti-FOXO1 antibody followed by immunoblotting with an anti-MDM2 antibody.

- 74 -

- - + + MG132 M HA M IP MW HA kD

IB: FOXO3A 78 FOXO3A

60

109 IB: MDM2 MDM2

78

60 Heavy chain

Figure 18 Interaction of endogenous MDM2 and FOXO3A in HEK293T cells. HEK 293T cells were treated with or without MG132 for 6 h before harvest. Cell extracts were immunoprecipitated with either an anti-HA antibody as control or an anti-MDM2 (SMP14) antibody followed by immunoblotting with an anti-FOXO3A antibody.

- 75 -

+--+- - FOXO1-WT --+-- + FOXO1-AAA --+++ - GST ++-- ++--- + GST-MDM2

GST Pull IB: FOXO1 Down

IB: FOXO1

CB: GST-MDM2

CB: GST

Figure 19 The interaction between FOXO1 and GST-MDM2 in vitro.

FOXO1 synthesized by in vitro transcription-translation reactions was incubated with GST-MDM2 fusion proteins, precipitated with glutathione beads and detected by immunoblotting with an anti-FOXO1 antibody. The amounts of GST proteins used in the pull-down assays were visualized by Coomassie blue staining after separation in a SDS-PAGE gel (bottom panel).

- 76 - included as a control. As shown in Figure 18, FOXO3A proteins were co-precipitated with MDM2 by an anti-MDM2 but not an anti-HA antibody. Treatment with MG132 increased the level of MDM2 and FOXO expression as well as the amount of proteins co-precipitated. These studies showed that co-precipitations occur with both endogenous and ectopically expressed proteins.

To determine whether MDM2 and FOXO1 interact in vitro, GST and GST-MDM2 fusion proteins were produced in bacteria, bound to glutathione beads and incubated with FOXO1 proteins produced by in vitro transcription coupled translations. In these

GST pull down assays, wild type FOXO1 was precipitated with GST-MDM2, but not with

GST (Figure 19). An active form of FOXO1, FOXO1 (AAA) in which all three AKT

phosphorylation sites were mutated to alanine, was also precipitated with GST-MDM2

but not with GST (Figure 19). This analysis demonstrated that FOXO1 and MDM2 form a

complex in vitro and that complex formation occurs independently of the phosphorylation

of FOXO1 by AKT.

3. The fork head box of FOXO and the region of MDM2 controlling nuclear-

cytoplasmic shuttling mediate the interaction between MDM2 and FOXO.

To define the region in FOXO1 that mediates the interaction with MDM2, H1299

cells were transfected with full length MDM2 and FOXO1 deletion constructs fused to the

HA tag (Figure 20a). Cellular extracts were subjected to co-immunoprecipitation with an

anti-MDM2 antibody. As shown in Figure 21, the full length FOXO1 protein was co-

precipitated with MDM2. Deletion of the C-terminal region of FOXO1 did not alter the

interaction whereas further deletion into fork head box abolished it, suggesting that the

fork head box is required for the interaction.

Further define the region in FOXO3A that mediates the interaction with MDM2,

purified bacteria expressed GST or GST fused fragments of FOXO3A (peptide P1-P5) - 77 - encoding five nonoverlapping FOXO3A regions (Figure 20b) were incubated with

HEK293T cell extracts overexpressed MDM2. MDM2 coprecipitated only with peptide P2

(amio acid 154-259), which contains the forkhead domain (Figure 22). These results suggest that MDM2 specifically interacts with the forkhead domain of FOXO3A. Further alignment the fork head domain of mouse FOXO3A, human FOXO1, FOXO3A and

FOXO4 (Figure 23), we found that the fork head domain is highly conserved in these four kinds of FOXO. All these data indicated MDM2 has a general effect on FOXO.

To define the region of MDM2 responsible for FOXO1 binding, different MDM2 constructs (Figure 24) were transfected into H1299 cells together with Flag-FOXO1.

Cellular extracts were immunoprecipitated with MDM2 antibody and the co-precipitated

FOXO1 was detected by anti-Flag antibody. As shown in Figure 25, all MDM2 mutants, except MDM2 (50-491) (p53 binding deficient) and MDM2 NLS-mt (182R, nuclear localization sequence defective), interacted with FOXO1. Immunoblot analysis showed that all MDM2 mutants were expressed at significant levels and most MDM2 proteins were detected in multiple forms, presumably due to cleavage by proteases (Chen et al.,

1997). In the presence of proteasome inhibitor MG132, however, both MDM2 (50-491) and MDM2 NLS-mt were co-precipitated with Flag-FOXO1 by the anti-Flag antibody

(Figure 26). The exact reason for the lack of interaction with FOXO1 in the absence of

MG132 is unclear, presumably due to the degradation of the protein complex by the proteasome.

Further analysis with additional MDM2 mutants in the presence of MG132, showed that the deletion of 150-230 amino acids abrogated the FOXO1 binding (Figure

27). Consistent with the fact that this region contains the nuclear localization sequence, it was also shown (Figure 28) that this mutant is mainly localized to cytoplasm. However, cytoplasmic localization is clearly not the reason for the lack of interaction with MDM2

- 78 - a 1 150 260 655 FK FL

1-150

1-270

256-655

1-655

b 1 148 258 672 GST FK FL

GST P1: 1-154

GST P2: 154-259

GST P3: 259-409

GST P4: 409-542

GST P5: 542-672

Figure 20 Diagram of different FOXO mutants used in this study.

a, The different mutants of human FOXO1 used in this study; b, The

different mutants of mouse FOXO3A used in this study.

- 79 -

HA-FOXO1 256-655 Vector 1-655 (FL) 1-655 1-150 1-270

1-655

IP: MDM2 IB: HA 1-270

IP: MDM2 IB: MDM2 MDM2 1-655

256-655 IB: HA 1-270

1-150

Figure 21 Mapping of the MDM2-interacting domain of FOXO1 by immunoprecipitation. H1299 cells were transfected with 2 µg MDM2 and 2 µg of different HA-tagged FOXO1 fragments. Anti-MDM2 immunoprecipitates were subjected to immunoblotting with anti-HA antibody and anti-MDM2 as indicated.

Immunoblotting of total cell extracts with anti-HA antibody (lower panel) showed the expression of different FOXO1 fragments.

- 80 -

GST P1 P2 P3 P4 P5 MDM2 + + + + + +

GST pull down MDM2

IB:MDM2

CB

GST fusion protein

Figure 22 Mapping of the MDM2-interacting domain of FOXO3A by

GST pull-down assay. Recombinant GST or GST fused with fragments of

FOXO3A (P1-P5) were amplified by bacteria expression and purified by

GST beads. HEK 293T cells were transfected with 4 µg MDM2. Cell lysates were subjected to GST pull down experiment. The amounts of GST proteins used in the pull-down assays were visualized by Coomassie blue (CB) staining after separation in a SDS-PAGE gel (bottom panel).

- 81 -

Figure 23 Alignment of FOXO member’s fork head box. Grey box

– fork head box; Green box –unique FOXO sequence.

- 82 -

P53 binding NLS NES Ring MDM2 0 100 200 300 400 491

1-361

50-491

Δ50-89

Δ89-150

Δ150-230

Δ222-325

Δ222-437

™ NES-mt

™ NLS-mt

™ E3-mt

™ 361D-A

Figure 24 Diagram of different MDM2 mutants used in the study.

- 83 - 222-437

MDM2 222-325 457S Δ 182R NES mutant 361D-A MDM2 50-491 Δ 1-361 Flag-FOXO1+ + + ++++++ +

FOXO1 IP: anti-MDM2 IB: anti-Flag

IB: anti-MDM2

MDM2 mutants

Figure 25 Mapping of the FOXO1-interacting domain in MDM2 by

coimmunoprecipitations in the absence of MG132. H1299 cells were

transfected with 2 μg Flag-FOXO1 and 2.5 µg different MDM2 fragments. Anti-

MDM2 mmunoprecipitates were probed with anti-Flag M2 antibody (upper

panel). Immunoblotting of cellular extracts with anti-MDM2 antibody (lower

panel) showed the expression of different MDM2 fragments.

- 84 - FL 50-491 5% input 5% MW FL 182R (kD)

MDM2/MDM2(182R) 75 MDM2(50-491) IP:M2 IB:MDM2 50

IB:M2 75 FOXO1

50

MDM2 IB:MDM2 75

50

Figure 26 An N-terminal truncation mutant and an NLS-mutant of MDM2 interact with FOXO1 in the presence of MG132. H1299 cells were transfected with 2 μg Flag-FOXO1 together with 5 μg MDM2 or 5 μg mutant

MDM2. Cells were treated with MG132 for 6 h before harvest. Cells lysate were immunoprecipitated with anti-Flag M2 antibody and detected with anti-MDM2 antibody. FL: full length MDM2, 50-491; MDM2(50-491); NLS: MDM2(NLS-mt).

- 85 - 89-150 150-230 50-89 Vector Δ Δ Δ 50-491 FL FL + - + + + + + Flag-FOXO1 MW (kD) + + + + + + + MG132

110 IP: M2 MDM2 78 IB: MDM2 60

170 110 IB: M2 78 FOXO1 60

47

170

IB: MDM2 110 MDM2 78

60

Figure 27 Truncation of the central region of MDM2 abolishes the interaction between MDM2 and FOXO1. H1299 cells were transfected with 2 μg

Flag-FOXO1 and 2.5 µg of different MDM2 fragments. Cells were treated with

MG132 for 6 h before harvesting. Anti-Flag immunoprecipitates were probed with anti-MDM2 (2A10) antibody (upper panel). Immunoblottiong of cellular extracts with anti-MDM2 antibody (lower panel) showed the expression of different MDM2 fragments.

- 86 -

MDM2 DAPI MDM2 DAPI

50-491 1-361

Δ50-89 NES-mt

Δ89-150 NLS-mt

E3-mt Δ150-230

361D-A Δ222-325

MDM2 Δ222-437

Figure 28 Immunofluorescence images show the cellular localization of various MDM2 mutants. H1299 cells were transiently transfected with MDM2 or different MDM2 mutants. 24 h posttransfection, cells were fixed and stained. The red signal showed the localization of MDM2 and the blue signal showed the nucleus of the cells.

- 87 - because co-precipitations were done with whole cell extracts and, under the same conditions, nuclear localization sequence mutant, the MDM2 NLS-mt, interacted with

FOXO1 (Figure 26). Besides cellular localization sequences, the 150-230 region also contains an inhibitory domain that suppresses cell cycle progression independently of p53. This region is also involved in interactions with several proteins, including TBP and p300.

4. MDM2 promotes the ubiquitination of FOXO1 and FOXO3A.

To test whether MDM2 promoted the ubiqutination of FOXO factors, H1299 cells were transfected with Flag-tagged FOXO1, wild type or an AKT phosphorylation site

FOXO1 mutant. The effect of ectopic MDM2 on FOXO1 ubiquitination by cotransfected

Myc-tagged ubiquitin was measured in the anti-Flag precipitates. In the absence of ectopic MDM2, limited FOXO1 ubiquitination was occured. This result is consistent with the fact that H1299 cells are p53-deficient and contain low levels of endogenous MDM2.

Cotransfection of MDM2 with increased the level of ubiquitiation of the wild type FOXO1 but not of FOXO1 (AAA). The data argue that the positive effect of MDM2 on FOXO1 ubiquitination requires phosphorylation at the AKT sites (Figure 29).

To test whether the effect of MDM2 extends to other FOXO factors, FOXO3A and His-tagged ubiquitin were transfected into H1299 cells and the effect of MDM2 on

FOXO3A ubiquitination was measured by immunblotting with anti-HA antibody following nickel bead pull-down under denaturing conditions. As shown in Figure 30, FOXO3A ubiquitination was increased by MDM2 in a manner dependent on the phosphorylation on the AKT sites. The data suggest that the stimulation of ubiquitination by MDM2 is not restricted to FOXO1 but among the FOXO factors.

In p53 and MDM2 double-null MEFs, wild type MDM2 stimulated the ubiquitination of FOXO1 in a dose dependent manner, whereas the MDM2 mutant - 88 - lacking the C-terminal ring finger E3 region did not exert such an effect (Figure 31), emphasizing the potential involvement of the E3 ligase activity. Nickel bead pull down assays under denaturing conditions revealed that MDM2 containing a point mutation in the E3 ligase domain, MDM2 E3 -mt (457S), did not stimulate FOXO1 ubiquitination

(Figure 32), confirming that FOXO1 ubiquitination by MDM2 requires its ubiquitin ligase activity. Interestingly, on the one hand, a constitutively nuclear MDM2 in which the nuclear export sequence was mutated, the NES-mt, did not promote FOXO1 ubiquitination (Figure 33), even though it contained an intact E3 ligase domain and interacted with FOXO1 (Figure 25). On the other hand, two cytoplasmic MDM2 mutants,

MDM2 NLS-mt and MDM2 (∆89-150), stimulated FOXO1 ubiquitination (Figure 32 and

33), showing that the ubiquitiantion of FOXO1 by MDM2 is likely to occur in the cytoplasm. MDM2 (∆150-230), which was located mainly in the cytoplasm (Figure 25) and did not interact with FOXO1 (Figure 26), was unable to stimulate FOXO1 ubiquitination, suggesting that the ubiquitination requires FOXO1 interaction. Overall, the data suggest that FOXO1 interacts with MDM2 in both the nucleus and the cytoplasm, but the ubiquitination by MDM2 requires the interaction in the cytoplasm.

To fully establish MDM2 as an E3 ligase for FOXO1 ubiquitination, the ability of recombinant MDM2 to catalyze the ubiquitination of FOXO1 was tested in vitro. In this experiment, GST-MDM2 stimulated the ubiquitination of in vitro translated FOXO1 in the

presence but not in the absence of purified E1, E2 and ubiquitin (Figure 34, upper

panel). GST fused to an MDM2 N-terminal fragment had no effect on the ubiquitination of

FOXO1. In reactions performed with transcription-coupled translation product from a control vector, the majority of the 35S-labeled ubiquitin conjugates disappeared, showing

that they were FOXO1 proteins (Figure 34, lower panel). In combination with the binding

- 89 -

- - + - +MDM2 + + + - - Flag-FOXO1wt MW (kD) - - - + + Flag-FOXO1 (AAA) - + + + + Myc-Ub

170

IP: M2 110 Ub-FOXO1 IB: myc 78

60

IB: M2 FOXO1

IB: MDM2 MDM2

Figure 29 MDM2 promotes the ubiquitination of FOXO1. H1299 cells were transfected with the indicated plasmids and cell extracts were either immunoprecipitated with M2 antibody, followed by immunoblotting with anti-Myc antibody, or directly immunoblotted with antibodies to Flag or MDM2.

- 90 -

- + + - - FOXO3Awt - - - + + FOXO3A (AAA) MW - - + - + MDM2 (kD) + + + + + His-Ub Ni-NTA pull down 170 Ub-FOXO3A 110 IB: FOXO3A 78 60

170

110 IB: MDM2 78 MDM2

60

Figure 30 MDM2 promotes the ubiquitination of FOXO3A. H1299 cells were transfected with the indicated plasmids and treated with MG132 for 6 h before harvest. Cell lysates were subjected to pull down analyses with Ni-NTA beads followed by immunoblotting with anti-FOXO3A antibody. Cell extracts were also subjected to direct immunoblotting by anti-MDM2 antibody.

- 91 -

- + + + + + Flag-FOXO1 + + + + + + Myc-Ub - - 2 4 - - MDM2 (μg) MW - - - - 2 4 MDM2 (1-361) (kD)

IP: M2 170 Ub-FOXO1 IB: Myc 110 78

MDM2 IB:MDM2

MDM2(1-361)

IB:M2 FOXO1

Figure 31 The MDM2 ring finger domain is critical for the ubiquination of

FOXO1. p53-/-,MDM2-/-MEF cells were transfected with the indicated plasmids including Myc-tagged ubiquitin and cell extracts were either immunoprecipitated with M2 antibody followed by immunoblotting with an anti-Myc antibody or immunoblotted directly with antibodies to Flag or MDM2.

- 92 - 222-325) 222-437) Δ Δ CTL MDM2( CTL MDM2(50-491) MDM2(NES mutant) MDM2(NLS mutant) MDM2 MDM2(457S) MDM2( MDM2(1-361) MDM2(361 mutant) FOXO1+ + + + + + + + + + - M.W. Ni-NTA (kD) pull down 170 Ub-FOXO1 IB:FOXO1 110 78

63

47

110

IB:MDM2 78 63 MDM2

47

IB:FOXO1 FOXO1

Figure 32 Ubiquitination of FOXO1 requires the MDM2 ubiquitin ligase function. P53-/-, MDM2-/- MEFs were transfected with Flag-FOXO1, His-

Ubiquitin and MDM2 mutants. Cell extracts were subjected to pull down assays with Ni-NTA beads followed by immunoblotting with anti-FOXO1 antibody.

- 93 -

89-150 50-89 150-230 FL Δ Vector Δ Δ MW (kD) Ni-NTA Pull Down 150 Ub-FOXO1 IB: FOXO1 100 75

IB: MDM2 MDM2

IB: FOXO1 FOXO1

Figure 33 The central region of MDM2 cannot promote the polyubiquitination of FOXO1. H1299 cells were transfected with 2 μg Flag-

FOXO1, 4 μg MDM2, and 4 μg His-ubiquitin. Cell extracts were subjected to pull down analyses with Ni-NTA beads followed by immunoblotting with anti-FOXO1 antibody.

- 94 -

+ + + FOXO1 + - - GST-MDM2-NT - + + GST-MDM2 MW (kD) + - + E1+E2+Ub

150 Ub-FOXO1

100 (35S labeled)

75

+ - FOXO1

MW + + GST-MDM2 (kD) + + E1+E2+Ub

Ub-FOXO1 150 (35S labeled) 100

75

Figure 34 MDM2 promotes FOXO1 polyubiquitination in vitro. GST-MDM2 and GST-N (containing MDM2 residues 1 to 150) were purified using glutathione agarose beads. Loaded beads were incubated with in vitro-translated FOXO1 in the presence or absence of E1 and E2 in an ubiquitination reaction as described in Materials and Methods. Polyubiquitinated FOXO1 appears as a high molecular weight smear above the unmodified FOXO1 band.

- 95 - data and whole-cell ubiquitination analysis with MDM2 mutants, the in vitro data

established that MDM2 functions as an ubiquitin E3 ligase for FOXO proteins.

5. MDM2 suppresses the expression of FOXO target genes and protects cells

from FOXO1-induced cell death.

FOXO target genes tumor necrosis related apoptosis inducing ligand (TRAIL), p27 CDK inhibitor and manganese superoxide dismutase (MnSOD) mediate the effect of

FOXO proteins on cell cycle arrest, apoptosis, and detoxification of reactive oxygen species. Their transcription products are directly regulated by FOXO factors. Consistent with the ubiquitination and degradation of FOXO factors by MDM2, stable expression of

MDM2 in H1299 cells decreased the level of TRAIL, p27 and MnSOD expression (Figure

35) whereas the knockdown of MDM2 increased the expression of TRAIL (Figure 36).

To test whether MDM2 protects cells from FOXO induced cell death, H1299 cells were transiently transfected with GFP and either control vector, FOXO1 or FOXO1 in combination with MDM2. The survival of the transfected cells was analyzed. The expression of FOXO1 decreased the number of transfected (green) cells, an effect that is relieved by MDM2 co-expression (Figure 37a). To confirm that the change in the viability of transfected cells is the result of cell apoptosis, transfected cells were fixed and stained with DAPI and their nuclear morphology was examined for features of apoptosis under a fluorescence microscope that allows the simultaneous visualization of blue and green fluorescence. Apoptotic index, as determined by scoring apoptotic cells in 300 green cells per sample, was 5% for controls and 25% for cells transfected with

FOXO1. Co-expression of MDM2 suppressed FOXO1-induced cell death in a dose- dependent manner (Figure 37b). The data were reproduced by independent

- 96 -

C E 1 V M - - 9 9 9 9 2 2 1 1 H H

MDM2 IB:MDM2

IB:FOXO3A FOXO3A

IB:FOXO1 FOXO1

IB:MnSOD MnSOD

IB:p27 p27

IB:TRAIL TRAIL

IB:CyclinD Cyclin D

IB:β-actin β-actin

Figure 35 Stable expressed MDM2 in H1299 cells regulates the expression of downstream targets of FOXO. Extracts of control (H1299-VEC) and MDM2 stable (H1299-M1) clones were subjected to immunoblotting with different antibodies as indicated in the figure.

- 97 -

L

siRNA T MDM2-1

C

IB:MDM2 MDM2

IB:FOXO3A FOXO3A

IB:TRAIL TRAIL

β IB:β-actin -actin

Figure 36 MDM2 siRNA increases the expression of the FOXO1 target gene

TRAIL. H1299 cells were transiently transfected with either GFP-siRNA or MDM2- siRNA. The cells were harvested at 48 h later. Cell extracts were subjected to immunoblotting analyses with anti-MDM2, anti-FOXO3A and anti-TRAIL antibodies.

- 98 - a 6h

9h

20h

pcDNA3 FOXO1 FOXO1+MDM2

b 30%

20%

10% index(%) Apoptosis 0% Flag-FOXO1 - + + +

MDM2(ug) 0 0 0.2 0.4

Figure 37 MDM2 promotes the cell survival in the presence of FOXO1.

- 99 - Figure 37 MDM2 promotes the cell survival in the presence of FOXO1. a, H1299 cells were transfected with pLNCE and Flag-FOXO1 in the presence or absence of MDM2. The viability of transfected cells in each well was scored by counting the number of green cells. Representative micrographs were captured by the fluorescence microscope that had a charge-coupled device camera. b, H1299 cells were transfected with the same plasmids as in a,

Apoptotic index of GFP-positive cells was determined by scoring 300 GFP- positive cells for chromatin condensation and nuclear fragmentation. Triplicate samples were analyzed per data point, and the graph represents three independent experiments.

- 100 -

a 40

30

20

Apoptosis (%) 10

0 - - + + Flag-FOXO1 - + - + MDM2 (μg)

40 30 20 10 Apoptosis (%) 0 - + + Flag-FOXO1 - - + MDM2 (μg)

Exp. No. Control MDM2 FOXO1 FOXO1+MDM2

1 10.7% 10.4% 32.4% 13.1% 2 16.7% - 49.0% 31.33%

Figure 38 MDM2 protects cells from FOXO1-induced cell death measured by Flow Cytometry.

- 101 -

b Control MDM2

4 4 10 0.6 1.1 10 1.72 2.12

103 103

102 102

7AAD 101 101 87.6 10.7 85.7 10.4

100 101 102 103 104 100 101 102 103 104

FOXO1 FOXO1+MDM2

104 4 1.52 4.13 10 1.18 2.84

103 103

102 102

101 101 62 32.4 82.9 13.1

100 101 102 103 104 100 101 102 103 104

Annexin-V APC

Figure 38 MDM2 protects cells from FOXO1-induced cell death measured by Flow Cytometry. H1299 cells were transfected with GFP-spectrin and Flag-

FOXO1 in the presence or absence of MDM2. The apoptotic GFP positive cells were detected with Annexin-V APC and 7-AAD. a, bar graphs; b,representative flow cytometry profiles.

- 102 - analysis of early apoptosis with the Annexin V method after FACS-based sorting. As shown in Figure 36, two independent analyses of cells cotransfected with GFP-spectrin showed that FOXO1 expression increased apoptosis in transfected cells by about 3-fold, which is partially suppressed by the co-tranefection of MDM2. The degree of induction by FOXO1 and the suppression by MDM2 varied between two experiments because the basal line of the FACS machine varied from time to time.

6. MDM2 transiently increased FOXO transcriptional activity.

Since FOXO factors are transcriptional factors, an important question which must be addressed is whether or not MDM2 affects the FOXO transcriptional activity. In order to answer this question, the following reporter genes were used in our experiment.

Synthetic reporter 3 × IRSLuc which contained three conserved insulin response sequence (IRS). FOXO1, like insulin, promotes the promoter activity through an IRS.

Cyclin D is the downstream target of FOXO factors in the cell cycle check point. FOXO

factors expression results in reduced levels of cyclin D protein expression. Cyclin D-Luc

is the reporter gene which can be used to specifically measure cyclin D activity in cells.

In order to measure the activity of the reporter gene, a transient transfection was

performed by transfecting the reporter gene, CMV-gal as an internal control, pcDNA3

control or Flag-FOXO1, and different doses of MDM2 into the DU145 cells and NIH3T3

cells. The data showed that MDM2 increased the 3 × IRSLuc activity, and this increase

occurred in a dose dependent manner (Figure 39 & Figure 40). FOXO1 decreased the

cyclin D-Luc activity and MDM2 decreased it further in a dose dependent manner

(Figure 41).

Our earlier data revealed that MDM2 interacted with both wild-type FOXO1 and

FOXO1 (AAA), however it only promoted the polyubiquitination of wild-type FOXO1.

- 103 -

45 40 MDM2-0ug 35 MDM2-0.1ug ) 5 30 MDM2-0.3ug 10

× 25 MDM2-0.5ug 20 (RLU, (RLU, IRSLuc activity activity IRSLuc 15 × 3 10 5 0 Endo-FOXO Flag-FOXO1wt

IB:M2

IB:MDM2

Figure 39 MDM2 increases the transcriptional activity of FOXO1 in a dose-dependent manner in DU145 cells. DU145 cells were transfected with

0.5 μg 3×IRSLuc, 0.1 μg CMV-Gal, 0.1 μg control vector or Flag-FOXO1, and different amounts of MDM2. FOXO activity was measured by the Promega luciferase activity kit.

- 104 -

16 14 NIH3T3 12 10 ) 5 8 10 × 6 4 (RLU, (RLU, IRSLuc activity activity IRSLuc

× 2 3 0

MDM2

Figure 40 MDM2 increases the transcriptional activity of FOXO1 in

NIH3T3 cells. NIH3T3 cells were transfected with 0.5 μg 3×IRSLuc, 0.1 μg

CMV-Gal, 0.1 μg Flag-FOXO1, and different amounts of MDM2. FOXO activity was measured by the Promega luciferase activity kit.

- 105 -

10 H1299 cells )

5 8 10 × 6

4 CyclinD Luc 2 activity (RLU, (RLU, activity

0

FOXO1 - + + + + MDM2 -

Figure 41 MDM2 enhances the ability of FOXO1 to inhibit cyclin D1.

H1299 cells were transfected with 0.5 μg cyclin D-Luc, 0.1 μg CMV-Gal, 0.1

μg Flag-FOXO1 and different amounts of MDM2. FOXO activity was measured by the Promega luciferase activity kit.

- 106 - a 30

25 MDM2-0ug MDM2-0.3ug ) 5 20

10 MDM2-0.5ug × 15

10 (RLU, (RLU, IRSLuc activity activity IRSLuc × 3 5

0 endo-FOXO1 FOXO1wt FOXO1 (AAA) b

30 MDM2-0ug ) 25 6

10 MDM2-0.3ug

× 20 MDM2-0.5ug 15 (RLU, (RLU, IRSLuc activity activity IRSLuc ×

3 10

5

0 FOXO1wt FOXO1(AAA)

Figure 42 MDM2 increases the transcriptional activity of both wild type

FOXO1 and the FOXO1(AAA) mutant in NIH3T3 and DU145 cells. Cells were transfected with 0.5 μg 3×IRSLuc, 0.1 μg CMV-gal, 0.1 μg Flag-FOXO1 or

FOXO1 (AAA) and different amounts of MDM2. FOXO activity was measured by the Promega luciferase activity kit. a, DU145; b, NIH3T3.

- 107 - MDM2 also increased the transcriptional activity of FOXO1 (AAA) (Figure 42). The data further indicated that MDM2 promotes FOXO transcriptional activity in an AKT- independent fashion.

7. p53 induces transient increase in the transcriptional activity of FOXO factors,

which is followed by FOXO degradation in an MDM2-dependent manner.

To test whether p53 affects the transcriptional activity of FOXO1, reporter gene 3

× IRSLuc, CMV-gal, pcDNA3 control or Flag-FOXO1 or Flag-FOXO1 (AAA), and different doses of p53 were transfected into DU145 cells (contain endogenous mutated p53) and LNCaP cells (contain endogenous wild type p53) and the transcriptional activity of FOXO1 was detemined. p53 decreased FOXO1 transcriptional activity in both cell lines (Figure 43a & Figure 44), but did not inhibit FOXO1-induced decrease in cell viability (Figure 43b). Introduction of MDM2 into the cells relieved the inhibition of FOXO activity by p53 (Figure 45).

In order to better understand the interaction among FOXO factors, p53 and

MDM2, H1299/V138 stable cell line that expresses a temperature-sensitive p53 was used. Shifting to a permissive temperature allows the activation of p53. At different time points after p53 activation, the transcriptional activity of endogenous FOXO factors and the level of FOXO3A and MDM2 protein expression were determined. Reporter analyses showed that FOXO activity was transiently induced between 5 and 24 hours after p53 activation and subsequently decreased (Figure 46a). Immunoblotting analysis showed that the MDM2 protein was induced to the highest level 5 hours after p53 activation follwed by gradually decrease of FOXO3A and MDM2 level (Figure 46b). MG132 treatment prevented the time-dependent decrease in FOXO3A levels (Figure 47), suggesting that the decrease is due to proteasome-mediated degradation. In order to

- 108 - a 120 100 p53-0ug ) 4 p53-0.02ug

10 80 × p53-0.1ug 60 p53-0.3ug (RLU, (RLU, 40 3XIRSLuc activity 3XIRSLuc activity 20 0 pcDNA3 FOXO1wt FOXO1(AAA) b

CTL FOXO1

FOXO1+p53 FOXO1+p53+MDM2

Figure 43 P53 inhibits the transcriptional activity of FOXO in a dose- dependent manner in DU145 cells, but not FOXO1-induced cell death. a, DU145 cells were transfected with 0.5 μg 3×IRSLuc, 0.1 μg CMV-Gal, 0.1 μg Flag-FOXO1 and different amounts of p53. FOXO activity was measured by the Promega luciferase activity kit. b, Cells were transfected with pLNCE and plasmids as described in panel a. Representative micrographs were captured by the fluorescence microscope that had a charge-coupled device camera.

- 109 -

10 p53-0ug 8

) p53-0.02ug 4

10 6 p53-0.1ug × 4 p53-0.3ug (RLU, (RLU,

3XIRSLuc activity 3XIRSLuc activity 2

0 pcDNA3 FOXO1wt FOXO1(AAA)

Figure 44 P53 inhibits the transcriptional activity of FOXO in a dose- dependent manner in LNCaP cells. DU145 cells were transfected with 0.5

μg 3×IRSLuc, 0.1 μg CMV-Gal, 0.1 μg Flag-FOXO1 and different amounts of p53. FOXO activity was measured by the Promega luciferase activity kit.

- 110 -

6 5 mtp53 ) 6 wtp53 4 10

× wtp53+MDM2 3 (RLU IRSLuc activity activity IRSLuc 2 × 3 1 0 FOXO1wt FOXO1(AAA)

Figure 45 MDM2 relieves the repression of FOXO1 activity by p53.

DU145 cells were transfected with 0.5 μg 3×IRSLuc, 0.1 μg CMV-gal, 0.1 μg

Flag-FOXO1, 0.1 μg HA-p53 and 0.5 μg of either control vector or MDM2.

FOXO activity was measured by luciferase activity kit (Promega).

- 111 - a 3 2.5 ) 5 2 10 × 1.5 (RLU, (RLU, IRSLuc activity activity IRSLuc 1 × 3 0.5 0 0h 1h 3h 5h 9h 24h 48h 72h

b 0 1 3 5 7 12 18 32 ºC (h)

IB:FOXO3A FOXO3A

IB:MDM2 MDM2

IB:p53 p53

Figure 46 MDM2 transiently increases FOXO transcriptional activity, which is followed by FOXO degradation . H1299/V138 cells cultured at 39oC were shifted to 32ºC for the indicated length of time. Cell extracts were prepared and assayed by luciferase assay (panel a) or by immunoblotting analyses (panel b).

- 112 -

- - - - + + + + MG132 0 5 9 18 0 5 9 18 32ºC (h)

150

100 IB: FOXO3A FOXO3A 75

50

IB: β-actin β-actin

Figure 47 MG132 relieves p53-induced decrease in the expression of

FOXO3A protein in H1299/V138 cells. Cells cultured at 39°C were shifted to

32ºC for indicated length of time. Cells were treated with MG132 for 6 h before cell extracts were prepared and subjected to immunoblot analysis.

- 113 -

Flag-FOXO1 - - + + + + MDM2 + - - - + + HA-p53 - + - + + -

IB:M2 FOXO1

p53 IB:HA

MDM2 IB:MDM2

IB: Tubulin Tubulin

Figure 48 p53 decreases the protein level of FOXO1 through MDM2.

H1299 cells were transiently transfected as noted on the Figure. 24h after transfection, cell lysates were subjected to immunoblotting with M2 antibody

(to detect FOXO1 expression), anti-HA antibody (to detect p53 expression), and anti-MDM2 (SMP14). Tubulin blots was included to show even loading in each sample.

- 114 -

- - + + GFP-siRNA + + - - MDM2-siRNA 37 32 37 32 Temperature (ºC)

IB: FOXO3A FOXO3A

IB:MDM2 MDM2

β-actin IB:β-actin

Figure 49 Knockdown of MDM2 partially relieves p53-induced FOXO3A downregulation. H1299/V138 cells were transfected with scrambled or MDM2 siRNA. 24 h posttransfection, cells were shifted to 32ºC and cultured for another 18 h. Then immunoblotting was performed with the indicated antibodies.

- 115 - determine which protein led to the degradation of FOXO factors, a transient transfection experiment was performed in H1299 cells. Immunoblotting analyses showed that p53 did not decrease the level of FOXO1 protein expression. MDM2, either alone or in combination with p53, decreased the level of FOXO1 protein expression (Figure 48).

Knockdown of MDM2 by siRNA partially relieved FOXO3A down-regulation by active p53, supporting the conclusion that the p53-induced decrease is MDM2-dependent

(Figure 49).

8. Site-specific sumoylation of SIRT1 regulates FOXO1 transcriptional activity

and stability.

Acetylation can affect protein degradation via ubiquitination (Ito et al., 2002;

Jeong et al., 2002; Li et al., 2002). The study in pancreatic β cells showed that FOXO1 acetylation inhibited the degradation of FOXO1 via the ubiquitin pathway (Kitamura et al., 2005).

SIRT1 is a mammalian HDAC. As a potential nutrient sensor, it regulates the lifespan of mammals in response to caloric restriction or nutrient starvation, and protects cells from apoptosis induced by DNA damage.

Previously (Brunet et al., 2004) and our studies (Figure 50) showed that SIRT1 inhibited FOXO1 transcriptional activity. After treating the H1299/V138 cells with SIRT1 inhibitor nicotinamide, we found that nicotinamide caused further increase of FOXO1 activity from 24 hours to 48 hours post p53 activiation (Figure 51). Therefore SIRT1 has a role in the action of p53-MDM2 on the activity and degradation of FOXO1.

Since SIRT1 affects the FOXO1 transcriptional activity and plays a potential role in FOXO1 stability, two very interesting questions arise: How does SIRT1 affect the transcriptional activity of FOXO1 and how is SIRT1 activity regulated. Our previous study

- 116 -

25

20 ) 5 10

× 15

10 (RLU IRSLuc activity activity IRSLuc × 3 5

0 123

FOXO1 - + + SIRT1 - - +

Figure 50 SIRT1 inhibits the transcriptional activity of FOXO. H1299 cells were transfected with 0.5 μg 3×IRS Luc, 0.1 μg CMV-Gal, 0.1 μg Flag-FOXO1 and

0.5 μg HA-SIRT1. FOXO activity was measured by the Promega luciferase activity kit.

- 117 -

18 H1299-V 16 H1299-V-NIC

) 14 5 H1299

10 12 × H1299-NIC 10 (RLU, (RLU, IRSLuc activity activity IRSLuc 8 × 3 6 4 2 0 0h 1h 3h 5h 8h 24h 48h

Figure 51 Nicotinamide treatment increases the FOXO transcriptional activity. H1299 and H1299/V138 cells were transfected with 0.5 μg 3×IRS

Luc and 0.1 μg CMV-Gal. 16 h later, the cells were treated with nicotinamide and transferred from 37°C to 32°C at indicated time points. FOXO activity was measured by the Promega luciferase activity kit.

- 118 -

Flag-FOXO1 + + + + HA-SIRT1 - - WT 734R HA-p300 - + + +

IB:Acetyl -K

IP:M2

IB:M2

p300 IB:HA

SIRT1 IB:HA

Figure 52 SIRT1 sumoylation at Lys 734 is required for FOXO1 deacetylation. H1299 cells were co-transfected with Flag-FOXO1, HA-p300 and either wild-type HA-SIRT1 (WT) or HA-SIRT1 mutated at Lys 734

(734R). Anti-Flag M2 immunoprecipitates were immunoblotted with antibody to acetylated FOXO1 or with an antibody to FOXO1. Cellular extracts were also immunoblotted with antibody to HA.

- 119 -

35 pcDNA3 ) 5 30 FOXO1

10 FOXO1+SIRT1 × 25 FOXO1+SIRT1(734R)

20

15

10

IRSLuc activity (RLU, (RLU, activity IRSLuc 5 × 3 0 12 CTL p300

Figure 53 Mutation of Lys 734 relieves the inhibition of FOXO1 transcriptional activity by SIRT1. The activity of transfected FOXO1 was measured using an IGFBP1 promoter-based reporter in H1299 cells expressing wild type SIRT1 or SIRT1 (734R) in the absence or presence of p300. CTL- control.

- 120 - showed that the sumoylation status of SIRT1 affects its deacetylase activity since a sumoylation site specific mutation abolished the deacetylase activity of SIRT1.

FOXO1 is a known substrate of SIRT1. In order to determine whether this sumoylation site affects the effect of SIRT1 on FOXO, Flag-tagged FOXO1 was cotransfected with p300, wild-type SIRT1 or SIRT1 (734R) in which the sumoylation site of SIRT1 was mutated, acetylated FOXO1 was detected by Western blotting of M2 immunoprecipitates with an antibody that recognizes acetylated FOXO1. As shown in

Figure 52, expression of wild type SIRT1 reduced amount of acetylated Flag-FOXO1 induced by p300, whereas expression of SIRT1 mutated at Lys 734 did not. When the cells transfected either FOXO1 with SIRT1, SIRT1 decreased FOXO1 transcriptional activity which is consistent with the previous study, whereas expression of SIRT1 mutated at Lys734 could not decrease the transcriptional activity of these two proteins

(Figure 53). These data showed that the sumoylation status of SIRT1 is critcal for its effect on FOXO1.

9. Genistein-induced FOXO1 expression is blocked by MDM2 expression in

H1299 cells.

Genistein is considered the primary anticancer component of soybeans. Its in vitro and/or in vivo activities include the antagonism of estrogen, inhibition of protein tyrosine phosphorylation, suppression of angiogenesis, inhibition of hydrogen peroxide formation induced by tumor promoters, inhibition of topoisomerases, induction of apoptosis and cell differentiation, scavenging of free radicals, and inhibition of carcinogenesis and tumor promotion. A previous paper showed that genistein down- regulated the MDM2 oncogene, induced apoptosis and inhibited proliferation in a variety

of human cancer cell lines, regardless of p53 status(Li et al., 2005). This raised an

- 121 -

Genistein (uM) 0 5 15 50 0 5 15 50

IB:FOXO1 FOXO1

β IB:β-actin -actin

IB:MDM2 MDM2

H1299-VEC H1299-M1

Figure 54 Genistein increases the expression of FOXO through MDM2 downregulation. H1299-VEC and H1299-M1 cells were plated in 100 mm dishes and treated with indicated doses of genistein for 24 h. Cell lysates were subjected to immunoblotting with anti-FOXO1 and anti-MDM2 antibodies.

- 122 - interesting question whether genistein’s anti-tumor activity is mediated through FOXO factors.

To address this question, parental H1299 cells and H1299 cells that stably express MDM2 (H1299-M) were plated in 100 mm dishes and treated with genistein at different doses. The expression of MDM2 and FOXO1 was determined by Western blot ananlysis. The analyses revealed that genistein decreased MDM2 and increased

FOXO1 level in a dose-dependent manner in H1299 parental cells. In H1299-M cells,

MDM2 expression was detected at a very high level. Genistein treatment had little effect on the expression of MDM2 or FOXO1 proteins (Figure 54). The data indicated that genistein increases FOXO1 level through MDM2 down regulation. It also indicated that FOXO factors may be one of the nuclear targets of genistein in suppressing tumorigenesis.

10. ARF promotes the MDM2-induced FOXO ubiquitination.

ARF is encoded by the INK4a-ARF and it is a known inhibitor of the MDM2

E3 ligase function on p53. To test whether ARF affects MDM2 E3 ligase function of

FOXO factors, H1299 cells were transfected with Flag-FOXO1, MDM2, myc-ARF and

His-ubiquitin as indicated in the figure 53. The cell lysates were purified by Ni-NTA

beads. The ubiquitinated-FOXO1 was detected by M2 anti-Flag antibody. As shown in

Figure 63, ARF increased FOXO1 ubiquitination level to a degree similar to that induced

by MDM2 (Figure 55). The combination of ARF and MDM2 increased the level of

FOXO1 ubiquitination to a degree comparable with that of MDM2 alone, suggesting that

AR may act through MDM2 to regulate FOXO ubiqutination (Figure 55). These data are

quite opposite to p53 ubiquitnation induced by MDM2 but are consistant with our

conclusion that FOXO ubiquitination occurs in the cytoplasm. It is known that ARF

inhibits the target of MDM2 to nucleoli to supprees the ubiquitination of p53. - 123 -

FOXO1 - + + + + MDM2 - - - + + Myc-ARF - - + - + His-Ubiquitin + + + + +

Ni-NTA pull down 170 Ub-FOXO1 110

IB:FOXO1 78

60

IB:myc 18 ARF 13 170 110 IB:M2 78 FOXO1 60

47

MDM2 IB:MDM2

Figure 55 ARF promotes the MDM2-induced FOXO1 ubiquitination. H1299 cells were transfected with 2μg Flag-FOXO1, 4 μg MDM2, 4 μg myc-ARF, 4 μg

His-ubiquitin as described on the Figure. Cell extracts were pull-down by Ni-NTA beads and the expression levels were detected by an anti-FOXO1 antibody.

- 124 -

DISCUSSION

Mammlian FOXO factors play very important roles in development, glucose metabolism, cancer, aging, and energy homeostatis. Several signaling pathways regulate their activity, such as the insulin pathway, glucose, IGF1 and oxidative stress. In this study, we found that MDM2 is the E3 ubiquitin ligase for FOXO factors. MDM2 interacts with FOXO factors and promotes their ubiquitination and degradation. It also affects their biological function and downstream targets expression of FOXO.

1. MDM2 is the E3 ubiquitin ligase of FOXO.

FOXO proteins are known to be ubiquitinated and their level of expression is regulated by proteasome mediated degradation (Aoki et al., 2004; Plas and Thompson,

2003), but a general ubiquitin E3 ligase for FOXO proteins remains to be identified.

Skp2 was reported to inhibit FOXO1 in tumor suppression through ubiquitin- mediated degradation but such activity appears to be restricted to FOXO1 (Huang et al.,

2005).

The present study identified MDM2 as a general ubiquitin E3 ligase for mammalian FOXO factors. We present multiple lines of evidences to support this conclusion. First, the manipulation of MDM2 expression by genetic deletion, knock down

MDM2 with siRNA or overexpression of MDM2 using transient or stable transfection leads to change of the level of FOXO1 and FOXO3A proteins in opposite directions.

Second, the down regulation of FOXO1 protein by MDM2 is relieved by treatment with a proteasome inhibitor, MG132, and the half-life of FOXO1 protein is decreased by ectopic - 125 - MDM2. Third, MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination.

The ubiquitination depends on both the ability of MDM2 to bind FOXO proteins as well as its E3 ligase activity. This is supported by the evidence that mutant MDM2 (∆150-230) that is missing in the FOXO binding region did not stimulate FOXO1 ubiquitination although it contains an intact E3 ring finger domain. Moreover, MDM2 mutants with either deletion {MDM2 (1-361)} or mutation {MDM2 E3-mt (457S)} at the C-terminus ring finger region lost their ability to stimulate FOXO ubquitination even though they are still able to interact with FOXO1. Finally, in test tube reaction, recombinant MDM2 catalyzed the ubiquitination of FOXO1, supporting a direct substrate-enzyme relationship.

2. MDM2-promoted ubiquitnation of FOXO depends on phosphorylation on PKB

sites.

In response to stimulation by IL-3, insulin or PDGF (platelet derived growth factor), the activated PKB oncogene triggered proteasome-dependent degradation of its substrates including FOXO1 and FOXO3A (Aoki et al., 2004; Matsuzaki et al., 2003;

Plas and Thompson, 2003). In our study, ubiquitination was found to depend on phosphorylation at sites that mediate the cytoplasmic localization of FOXO factors by

PKB. FOXO1 (AAA), in which all three PKB sites are mutated to non-phosphorylatable alanine, did bind to MDM2 (Figure 19) but its level of ubiquitination was not affected by

MDM2 (Figure 29). Similarly, ubiquitination of FOXO3A (AAA) was unaffected by MDM2

(Figure 30). These data suggest that MDM2 acts as a conditional E3 ligase for FOXO proteins, which is only functional after FOXO phosphorylation at AKT sites. Previous studies (Feng et al., 2004; Zhou et al., 2001) showed that phosphorylation of MDM2 by

PKB inhibits its interaction with ARF, thereby increasing its stability and promoting its ability to bind and degrade p53. It remains to be determined whether the MDM2 phosphorylation active PKB regulates its ability to stimulate FOXO ubiquitination. - 126 - 3. MDM2-promoted ubiqutination of FOXO happens mainly in the cytoplasm.

Protein ubiquitination and degradation occur in both the nucleus and cytoplasm

(Yu et al., 2000). In our analysis, the nuclear MDM2 in which the nuclear export sequence is mutated contains an intact E3 region and is able to bind FOXO1 but unable to increase FOXO1 ubiquitination, on the other hand, the cytoplasmic MDM2, both the

MDM2 NLS-mt and MDM2 (∆89-150), stimulated ubiquitination, suggesting that it is the cytoplasmic MDM2 that functions as an E3 ligase for FOXO factors. In combination with the data that showed that nuclear FOXO1 (AAA) is not ubiquitinated by MDM2, it is reasonable to assume that FOXO ubiquitination occurs in the cytoplasm. However, such a conclusion might seem incompatible with the immunofluorescence staining data showing that the colocalization of MDM2 and FOXO occured mainly in the nucleus. It should be pointed out that the colcoalization studies were performed in cells grown in a medium containing low levels of serum, which minimizes PKB activity and inhibits FOXO cytoplasmic localization and degradation. Consistent with the degradation in the cytoplasm, the interaction between cytoplasmic MDM2 and FOXO1 was difficult to detect unless proteasome activity was inhibited by MG132. The fact that MDM2 NLS-mt that cannot enter the nucleus interacting with FOXO1 and stimulating its ubiquitination suggests that a nuclear interaction between MDM2 and FOXO proteins is not required for FOXO ubiquitination. Presently, the details of the nuclear interaction between MDM2 and FOXO proteins are unclear. It is possible that nuclear interaction between FOXO factors and MDM2 may exert an effect on FOXO factors that is distinct from degradation.

In the case of p53 ubiquitination, it has been shown that monoubiquitination by MDM2 promotes the cytoplasmic localization of p53 whereas the polyubiquintination stimulates the degradation of p53 in nucleus (Li et al., 2003a).

- 127 - 4. MDM2 affects both the transcriptional activity and ubiquitination of FOXO.

FOXO factors are transcription factors. On one hand, FOXO1, FOXO3A and

FOXO4 directly bind to the promoters of TRAIL, Bim, p27, MnSOD, GADD45, G6Pase,

PEPCK, TAT and IGFBP-1 and of others. On the other hand, FOXO can also indirectly regulate other genes, such as cyclin D1. In our studies, a positive effect of MDM2 on the transcriptional activity of FOXO1 was detected in transient transfection reporter assays.

It also promoted the ubiquitination and degradation of FOXO factors, leading to FOXO protein level decreases. It is not fully understood as to how MDM2 decreases FOXO proteins expression level. There are three possible mechanisms that might be responsible for MDM2’s positive effect on transcriptional activity of FOXO: 1) MDM2 promotes the transcriptional activity first, followed by its role in FOXO degradation because transcriptional activity data in H1299/V138 cells showed that FOXO activity was transiently induced between 5h and 48 h post p53 activation and subsequently FOXO activity decreased (Figure 47a); 2) FOXO4 was recently shown to be monoubiquitinated, which increases its transcriptional activity (van der Horst et al., 2006) and this raises the second possibility. MDM2 plays two roles in the regulation of FOXO factor. It may promote FOXO transcriptional activity through monoubiquitnation but degrade FOXO protein through polyubiquitnation; 3) About 20 proteins are reported to interact with

MDM2, and p300 is reported to play a critical role in the MDM2-directed turnover of p53

(Grossman et al., 1998) and this raises the third possibility. Different cofactors are involved in the regulation of FOXO factors by MDM2 leading to different outcomes.

In our p53 study, Mdm2-mediated monoubiquitylation of p53 greatly promoted its mitochondrial translocation and thus its apoptosis in the mitochondria. Upon entrance in the mitochondria, p53 undergoes rapid deubiquitylation by mitochondrial HAUSP via a stress-induced mitochondrial p53-HAUSP complex (Marchenko et al., 2007). FOXO4

- 128 - was also reported to become monoubiquitinated in response to increased cellular oxidative stress, resulting in its re-localization to the nucleus and an increase in its transcriptional activity. Deubiquitination of FOXO is catalyzed by the HAUSP (herpesvirus-associated ubiquitin-specific protease), which interacts with and deubiquitinates FOXO in response to oxidative stress. We found that MDM2 suppresses the expression of FOXO1 target genes and protects cells from FOXO1- induced apoptosis. These observations are consistent with the effect of MDM2 on the translocation change and degradation of FOXO facotors. Further study is required to understand the biological effect of MDM2 promoted transcriptional activity of FOXO factors and to estabolish whether FOXO factors have any mitochondrial function.

5. Mammalian FOXO factors interact with p53.

Mammalian FOXO factors and p53 are tumor suppressors and the regulators of aging that act similarly in many ways. They induce apoptosis (Modur et al., 2002) and cell cycle arrest (Medema et al., 2000; Nakamura et al., 2000; Schmidt et al., 2002) and regulate cellular responses to DNA damages and stress (Essers et al., 2004; Kajihara et al., 2006) through transcriptional induction of a similar set of target genes such as the p21 CDK inhibitor, Fas ligand and GADD45. In our study, we found that ectopically expressed p53 inhibited FOXO transcriptional activity in either DU145 cells (contain inactive p53) or LNCaP (contain wild type p53) but did not decrease FOXO1 induced cell death (Figure 44b). Two subsequent papers showed that p53 interacted with FOXO3A in the presence of stress and active FOXO3A could induce p53-dependent apoptosis and promote p53 cytoplasmic accumulation by increasing its association with the nuclear exporting machinery. All these evidence indicate that the regulation of transcriptional activity of p53 and FOXO factor is independent of the regulation of the proapoptotic activity of p53 and of FOXO factors. - 129 - p53 and MDM2 form a feedback loop, in which p53 induces MDM2 by activating

MDM2 transcription, and MDM2 in turn negatively regulates p53 by binding and promoting p53 ubiquitination and degradation (Jin and Levine, 2001). This feedback loop keeps the p53 activity in check under normal conditions. Upon DNA damages, the interaction between MDM2 and p53 is suppressed, resulting in an increased p53 activity that triggers apoptosis. Our data suggest that MDM2 might act as a general coordinator to turn off multiple negative growth regulators when p53 senses that the DNA damage has been repaired (Figure 56). During our investigations, several studies documented a functional interaction between p53 and FOXO3A. In the presence of hydrogen peroxide, p53 and FOXO3A form a complex (Brunet et al., 2004). DNA damage promotes

FOXO3A nuclear export through p53-dependent activation of serum and glucocorticoid activated kinases (You et al., 2004a). In endothelial cells (Miyauchi et al., 2004) and dermal fibroblasts (Kyoung Kim et al., 2005), inhibition of FOXO3A by PKB or siRNA promoted senescence-like growth arrest in a p53- and p21-dependent manner. Overall, p53 and FOXO factors appear to have a complex relationship, the outcome of which is likely to depend on cellular status and environment.

6. MDM2 regulates FOXO factors in a p53 independent way.

In addition to the negative regulation of p53 activity, MDM2 is overexpressed in tumor cells and functions as an oncogene to promote cancer cell growth independent of p53 (Daujat et al., 2001; Ganguli and Wasylyk, 2003). Activated FOXO3A was also reported to impair the transcriptional activity of p53, but enhanced its pro-apoptotic function in mitochondria (You et al., 2006b). The present studies suggest that suppression of FOXO factors may be one of the p53-independent mechanisms by which

MDM2 promotes cancer cell survival and cell growth. Decreased FOXO3A and FOXO1

- 130 -

MDM2 Overexpression in Tumor Cells

MDM2 E3 Ligase

Stimuli Oxidative stress, p53 Tumor Suppression DNA damages, Apoptosis, & therapeutic Senescence, treatments cell cycle arrests, & DNA repair FOXO

Ub Ub FOXO Ub

Ub Proteasome p53 Ub Ub

Degradation

Figure 56 A working model shows the functional interaction among FOXO, p53 and MDM2 .

- 131 - activity from loss of PTEN led to a decrease in TRAIL expression and increased survival of prostatic tumor cells (Modur et al., 2002). Loss of p27 expression is associated with aggressive behavior in a variety of human epithelial tumors (Catzavelos et al., 1997;

Macri and Loda, 1998). Stable expression of MDM2 in H1299 cells decreased the expression of FOXO3A together with TRAIL, p27 and MnSOD (Figure 35). Knockdown of MDM2 by siRNA in H1299 cells increased the level of FOXO3A together with TRAIL

(Figure 36). These results suggest that MDM2 promotes tumorigenesis through down- regulation of FOXO target genes and that the targeted interruption of FOXO ubiquitination and degradation by MDM2 may represent an effective strategy for cancer prevention and therapy.

7. Different domains of MDM2 play different roles in the regulation of p53 and

FOXO factors (see Table 2).

FOXO factor and p53 are the central regulators of cellular reponse to stress, genotoxic insult and DNA damage. MDM2 interacts with and promotes the degradation of p53. Our study shows that MDM2 also interacts with and promotes the ubiquitination and degradation of FOXO. But it became quite clear that different domains of MDM2 play different roles in the regulation of these two proteins. MDM2 (50-491), MDM2 (Δ50-

89) and MDM2 (Δ 90-150) cannot bind with p53 and promotes its ubiquitination, but they do bind with FOXO and can promote the ubquitination of FOXO factors. MDM2 (Δ 150-

230) binds with p53 and promotes the ubiquitinaion of p53 but it does not interact with

FOXO and promotes the ubiquitnation of FOXO. The nucleo-cytoplasmic shuttling is critical for MDM2-promoted ubiquitination of p53 because even MDM2 (NES mutant) and MDM2 (NLS mutant) are capable of binding to p53, but cannot promote its

- 132 - Binds Increases Binds Promotes the Loca to ubiquitinatio to ubquitination of lizati FOXO1 nof FOXO1 p53 p53 on

MDM2 + + + + N (Haupt,1997) MDM2(1-361) + - + - N (Pochampally,1999) MDM2(50-491) + + - ND N MDM2(Δ50-89) + + - ND N MDM2(Δ 90-150) ± + - ND C/N MDM2(Δ 150-230) - - + + C (Dai, 2004) MDM2(NES mutant) + - + - N (Roth,1998) MDM2(NLS mutant) + + + - C (Tao, 1999) MDM2(Δ 222-325) + - + ND N MDM2(Δ 222-437) + + + - N (Kubbutat,1998) MDM2(457S) + - + - N (Pan,2003) MDM2(361D-A) + - + + N (Pochampally,1999)

Table 2 Localization and function of different MDM2 mutants. ND- Not

done yet ; N-nucleus; C-cytoplasm.

- 133 - ubiquination. MDM2 (Δ 222-437) is capable of binding to p53 and FOXO; as for ubiquitnaiton, this mutant can only promote the ubiquitnation of FOXO but not p53. One possible reason for this effect is the nucleolar localization of MDM2 (Δ 222-437). MDM2

(1-361) is a mutant which is deleted of the two highly conserved RING finger motifs in the C-terminus. MDM2 (457S) carries a mutation critical for E3 function of MDM. It prevents the polyubiquitination of p53, significantly lowers the efficiency of MDM2 interaction with MDMX and promotes MDMX polyubiquitination. Both of these mutants can bind to p53 and FOXO factors but cannot promote their ubiquitination. This indicates the essential role of the E3 region of MDM2 in the ubiquitination of both p53 and FOXO.

8. The sumoylation status of SIRT1 affects the stability and activity of FOXO.

Acetylation affects protein degradation via ubiquitination pathway (Ito et al., 2002;

Jeong et al., 2007; Li et al., 2002). In β-cells, acetylation of FOXO1 decreased its degradation via the ubiquitin pathway (Kitamura et al., 2005). The current study showed that sumoylation of wild type SIRT1 increased its deacetylase activity,wild type SIRT1, but not SIRT1 (734R), decreased the acetylation level and transcriptional activity of

FOXO1. This study suggests that sumoylation of SIRT1 affects its ability to regulate the transcriptional activity of FOXO1. The study also raised the possibility that SIRT1 sumoylation may regulate the stability of FOXO1.

9. Genistein increases FOXO expression level through down-regulation of MDM2.

Genistein (5,7,4'-trihydroxyisoflavone), a kind of isoflavone, is now considered to be the primary anticancer component of soybeans. Experimental data showed that in

H1299 lung cancer cells, genistein increases the FOXO1 protein expression level in a dose dependent manner, overexpression of MDM2 abolishes the effect of genestein on

FOXO1 (Figure 54). The data indicates that genstein increases FOXO1 protein

- 134 - expression level and this effect is MDM2-dependent. Previous data showed that genistein down-regulates MDM2 expression at both the transcriptional and posttranslational levels, independently of p53, in both human cancer cell lines and primary cells. Up-regulateion of the tumor suppressor p21Waf1/CIP1 by genistein can be

regulated by both p53 and FOXO factors. Thus, the inhibition of MDM2 expression by

genistein may be essential for its antitumor activities.

10. ARF differentially affects E3 function of MDM2 toward different substrates.

p19ARF is the product of an alternative open reading frame of the mouse INK4a-

ARF locus. It is a known fact that ARF is an inhibitor of the MDM2 E3 ligase function on

p53 (Zhang and Xiong, 2001). In our study, also it was found that ARF stimulated

FOXO1 ubiqutination as well (Figure 55). The data indicate that ARF interaction with

MDM2 differentially affects its E3 function toward different substrates rather than

inactivating its E3 function in general. This effect is plausible because ARF does not

directly interact with the RING domain of MDM2, which may be involved in recruiting E2.

Binding of ARF to the acidic domain of MDM2 may simply alter its ability to properly

orient E2 for the transfer of ubiquitin to certain substrates. In the case of p53, ubiquitin conjugation is blocked. In the case of FOXO1, ubiquitination is stimulated. There are two

possible explanations for this. The first one is that ARF may stablize MDM2 interaction

with FOXO, which may account for increased FOXO1 ubiqutination. The second one is

that ARF may also qualitatively stimulate the E3 activity of MDM2 toward FOXO. Further

experiments using the in vitro ubiquitination system will be required to address this

matter.

- 135 -

SUMMARY AND PERSPECTIVES

Cancer is a major health problem in the USA and worldwide. One third of the people suffer from some form of cancer and 20% of all deaths are cancer related. In developed countries, cancer care represents about 10% of total health costs. Since the incidence of cancer increases with age and people live longer, some important questions need to be addressed: Why is cancer more prominent in older people and should cancer treatment be different for the various age groups. Our studies focus on the regulation of

FOXO factors which are key elements of tumor initiation and of the mechanisms that regulate an ’s lifespan. They are potential candidates to serve as molecular linker between longevity and cancer. FOXO factors also regulate numerous cellular processes, such as stress resistance, the cell cycle, apoptosis, DNA repair/metabolism and tumorigenesis

Ubiqitination controls the stability/degradation of FOXO factors, although the mechanism is not clearly defined. Our data allow us to draw the following conclusions:

(1) MDM2 binds directly to FOXO1. Specifically, the binding between MDM2 and FOXO1 is through the central region of MDM2 and the N-terminal region of FOXO1.

Interestingly, FOXO protein phosphorylated by AKT is not required for this binding; (2)

MDM2 binding promotes the ubiquitin-dependent degradation of FOXO1 and FOXO3A.

Knockdown of MDM2 by siRNA caused accumulation of both FOXO1 and FOXO3A.

MDM2 mediated polyubiquitination of FOXO appears to occur in the cytoplasm and is an

AKT phosphorylation dependent; (3) Recombinant MDM2 catalyzes the ubiquitination of

- 136 - FOXO1 in test tube reactions, demonstrating a direct substrate-enzyme relationship; (4)

MDM2 affects the expression of downstream FOXO targets and protects cells from

FOXO1 induced cell death; (5) p53 transiently induced the transcription activity of

FOXO3A, followed by degradation of FOXO3A protein through MDM2.

In addition to the above conclusions, we also made several preliminary but potentially important observations that need to be investigated in further studies.

1) In the transient transfection experiments, MDM2 increased the transcriptional activity of both wild type FOXO factors and FOXO (AAA), which is no longer phosphorylated by AKT anymore. The molecular mechanism underlying this effect needs further clarification and monoubiquitination could play a decisive role. Other important questions to be addressed are: whether monoubiquiyination of FOXO factors is induced by MDM2 and is yes, whether monoubiquitination and polyubiquitination play different roles in FOXO function; whether monoubiquitination increase the transcriptional activity of FOXO without an effect on protein stability; whether MDM2 levels has a differential effect on the ubiqutination status of FOXO; and whether cofactors, such as p300 and SIRT1, have any effect on the ubiqutination status of FOXO1.

2) Genistein increases the expression of FOXO1 protein in a dose dependent manner in H1299 lung cancer cells. This effect is abolished in the H1299 cells that stably express MDM2. Our data suggested that the anti-tumor effect of genistein may be mediated, at least in part, through the down-regulation of MDM2, causing the up- regulation of FOXO factors (our present data) and p53 (earlier data of others). It remains to be seen whether this effect of genistein on FOXO1 can be extended to other FOXO factors and other cells lines and if so, whether it only affects FOXO protein stability or through other mechanisms. It is also important to determine how genistein may affect downstream targets of FOXO factors.

- 137 - 3) It is an estabolished fact that PKB and insulin promote FOXO ubiquitination and degradation. It remains to be elucidated whether MDM2 is required for the PKB and insulin promoted degradation of FOXO factors and whether the growth factors control the levels of mono- vs. poly-ubiquitination.

4) SIRT1 acts as a “double-edged sword” promoting survival of aging cells, but also increasing the cancer risk. Several studies had shown that acetylation inhibits

FOXO protein degradation. One key question is whether SIRT1 affects MDM2 promoted ubiquitination and degradation, and if so, whether sumoylation mutant of SIRT1 will affect stability of FOXO since the mutant is associated with a decrease in its deacetylase activity and with reduced ability to inhibit the transcriptional activity of FOXO1.

5) ARF is known to inhibit MDM2-induced p53 ubiquitination, but enhance

MDM2-induced MDMX ubiquitination. In our study, ARF promoted FOXO ubiquitination.

It is very important to define the molecular mechanism behind the differential effect of

ARF on the MDM2-induced ubiquitination of p53 and FOXO factors.

In summary, my thesis work is the first to identify MDM2 as an E3 ubiquitin ligase for FOXO factors and suggests that the targeted inhibition of MDM2 E3 activity may be a more effective strategy for tumor supprerssion than the current strategy that targets solely the interaction with p53.

- 138 -

REFERENCES

Agami, R., Blandino, G., Oren, M., and Shaul, Y. (1999). Interaction of c-Abl and p73alpha and their collaboration to induce apoptosis. Nature 399, 809-813.

Alkhalaf, M., Ganguli, G., Messaddeq, N., Le Meur, M., and Wasylyk, B. (1999). MDM2 overexpression generates a skin phenotype in both wild type and p53 null mice. Oncogene 18, 1419-1434.

Alkhateeb, A., Fain, P.R., and Spritz, R.A. (2005). Candidate functional promoter variant in the FOXD3 melanoblast developmental regulator gene in autosomal dominant vitiligo. The Journal of investigative dermatology 125, 388-391.

Alvarado-Sanchez, B., Hernandez-Castro, B., Portales-Perez, D., Baranda, L., Layseca- Espinosa, E., Abud-Mendoza, C., Cubillas-Tejeda, A.C., and Gonzalez-Amaro, R. (2006). Regulatory T cells in patients with systemic lupus erythematosus. Journal of autoimmunity 27, 110-118.

Amerik, A.Y., Li, S.J., and Hochstrasser, M. (2000). Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae. Biological chemistry 381, 981-992.

Anekonda, T.S., and Reddy, P.H. (2006). Neuronal protection by sirtuins in Alzheimer's disease. Journal of neurochemistry 96, 305-313.

Aoki, M., Jiang, H., and Vogt, P.K. (2004). Proteasomal degradation of the FoxO1 transcriptional regulator in cells transformed by the P3k and Akt oncoproteins. Proceedings of the National Academy of Sciences of the United States of America 101, 13613-13617.

Apionishev, S., Malhotra, D., Raghavachari, S., Tanda, S., and Rasooly, R.S. (2001). The Drosophila UBC9 homologue lesswright mediates the disjunction of homologues in meiosis I. Genes Cells 6, 215-224.

Arden, K.C. (2006). Multiple roles of FOXO transcription factors in mammalian cells point to multiple roles in cancer. Experimental gerontology 41, 709-717.

Armoni, M., Harel, C., Karni, S., Chen, H., Bar-Yoseph, F., Ver, M.R., Quon, M.J., and Karnieli, E. (2006). FOXO1 represses peroxisome proliferator-activated receptor- - 139 - gamma1 and -gamma2 gene promoters in primary adipocytes. A novel paradigm to increase insulin sensitivity. The Journal of biological chemistry 281, 19881-19891.

Aza-Blanc, P., Di Lauro, R., and Santisteban, P. (1993). Identification of a cis-regulatory element and a thyroid-specific nuclear factor mediating the hormonal regulation of rat thyroid peroxidase promoter activity. Molecular endocrinology (Baltimore, Md 7, 1297- 1306.

Bailey, D., and O'Hare, P. (2002). Herpes simplex virus 1 ICP0 co-localizes with a SUMO-specific protease. The Journal of general virology 83, 2951-2964.

Bakker, W.J., Blazquez-Domingo, M., Kolbus, A., Besooyen, J., Steinlein, P., Beug, H., Coffer, P.J., Lowenberg, B., von Lindern, M., and van Dijk, T.B. (2004). FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1. The Journal of cell biology 164, 175-184.

Balaban, R.S., Nemoto, S., and Finkel, T. (2005). Mitochondria, oxidants, and aging. Cell 120, 483-495.

Balciunaite, G., Keller, M.P., Balciunaite, E., Piali, L., Zuklys, S., Mathieu, Y.D., Gill, J., Boyd, R., Sussman, D.J., and Hollander, G.A. (2002). Wnt glycoproteins regulate the expression of FoxN1, the gene defective in nude mice. Nature immunology 3, 1102- 1108.

Bandyopadhyay, D., Okan, N.A., Bales, E., Nascimento, L., Cole, P.A., and Medrano, E.E. (2002). Down-regulation of p300/CBP histone acetyltransferase activates a senescence checkpoint in human melanocytes. Cancer research 62, 6231-6239.

Banerjee, A.C., Recupero, A.J., Mal, A., Piotrkowski, A.M., Wang, D.M., and Harter, M.L. (1994). The adenovirus E1A 289R and 243R proteins inhibit the phosphorylation of p300. Oncogene 9, 1733-1737.

Bannister, A.J., and Kouzarides, T. (1996). The CBP co-activator is a histone acetyltransferase. Nature 384, 641-643.

Barak, Y., Juven, T., Haffner, R., and Oren, M. (1993). mdm2 expression is induced by wild type p53 activity. The EMBO journal 12, 461-468.

Bartel, F., Taubert, H., and Harris, L.C. (2002). Alternative and aberrant splicing of MDM2 mRNA in human cancer. Cancer cell 2, 9-15.

Baumeister, R., Schaffitzel, E., and Hertweck, M. (2006). Endocrine signaling in Caenorhabditis elegans controls stress response and longevity. The Journal of endocrinology 190, 191-202.

- 140 - Bedalov, A., Gatbonton, T., Irvine, W.P., Gottschling, D.E., and Simon, J.A. (2001). Identification of a small molecule inhibitor of Sir2p. Proceedings of the National Academy of Sciences of the United States of America 98, 15113-15118.

Bennett, C.L., Christie, J., Ramsdell, F., Brunkow, M.E., Ferguson, P.J., Whitesell, L., Kelly, T.E., Saulsbury, F.T., Chance, P.F., and Ochs, H.D. (2001). The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nature genetics 27, 20-21.

Bennin, D.A., Don, A.S., Brake, T., McKenzie, J.L., Rosenbaum, H., Ortiz, L., DePaoli- Roach, A.A., and Horne, M.C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G1/S phase cell cycle arrest. The Journal of biological chemistry 277, 27449- 27467.

Best, J.L., Ganiatsas, S., Agarwal, S., Changou, A., Salomoni, P., Shirihai, O., Meluh, P.B., Pandolfi, P.P., and Zon, L.I. (2002). SUMO-1 protease-1 regulates gene transcription through PML. Molecular cell 10, 843-855.

Bieller, A., Pasche, B., Frank, S., Glaser, B., Kunz, J., Witt, K., and Zoll, B. (2001). Isolation and characterization of the human forkhead gene FOXQ1. DNA and cell biology 20, 555-561.

Blixt, A., Landgren, H., Johansson, B.R., and Carlsson, P. (2007). Foxe3 is required for morphogenesis and differentiation of the anterior segment of the eye and is sensitive to Pax6 gene dosage. Developmental biology 302, 218-229.

Bluher, M., Kahn, B.B., and Kahn, C.R. (2003). Extended longevity in mice lacking the insulin receptor in adipose tissue. Science 299, 572-574.

Bordone, L., Motta, M.C., Picard, F., Robinson, A., Jhala, U.S., Apfeld, J., McDonagh, T., Lemieux, M., McBurney, M., Szilvasi, A., Easlon, E.J., Lin, S.J., and Guarente, L. (2006). Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells. PLoS biology 4, e31.

Borrow, J., Stanton, V.P., Jr., Andresen, J.M., Becher, R., Behm, F.G., Chaganti, R.S., Civin, C.I., Disteche, C., Dube, I., Frischauf, A.M., Horsman, D., Mitelman, F., Volinia, S., Watmore, A.E., and Housman, D.E. (1996). The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein. Nature genetics 14, 33-41.

Bouras, T., Fu, M., Sauve, A.A., Wang, F., Quong, A.A., Perkins, N.D., Hay, R.T., Gu, W., and Pestell, R.G. (2005). SIRT1 deacetylation and repression of p300 involves lysine residues 1020/1024 within the cell cycle regulatory domain 1. The Journal of biological chemistry 280, 10264-10276.

- 141 - Boyd, S.D., Tsai, K.Y., and Jacks, T. (2000). An intact HDM2 RING-finger domain is required for nuclear exclusion of p53. Nature cell biology 2, 563-568.

Brownawell, A.M., Kops, G.J., Macara, I.G., and Burgering, B.M. (2001). Inhibition of nuclear import by protein kinase B (Akt) regulates the subcellular distribution and activity of the forkhead transcription factor AFX. Molecular and cellular biology 21, 3534-3546.

Brunet, A., Bonni, A., Zigmond, M.J., Lin, M.Z., Juo, P., Hu, L.S., Anderson, M.J., Arden, K.C., Blenis, J., and Greenberg, M.E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857-868.

Brunet, A., Park, J., Tran, H., Hu, L.S., Hemmings, B.A., and Greenberg, M.E. (2001). Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Molecular and cellular biology 21, 952-965.

Brunet, A., Sweeney, L.B., Sturgill, J.F., Chua, K.F., Greer, P.L., Lin, Y., Tran, H., Ross, S.E., Mostoslavsky, R., Cohen, H.Y., Hu, L.S., Cheng, H.L., Jedrychowski, M.P., Gygi, S.P., Sinclair, D.A., Alt, F.W., and Greenberg, M.E. (2004). Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303, 2011-2015.

Brunkow, M.E., Jeffery, E.W., Hjerrild, K.A., Paeper, B., Clark, L.B., Yasayko, S.A., Wilkinson, J.E., Galas, D., Ziegler, S.F., and Ramsdell, F. (2001). Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nature genetics 27, 68-73.

Buschmann, T., Lerner, D., Lee, C.G., and Ronai, Z. (2001). The Mdm-2 amino terminus is required for Mdm2 binding and SUMO-1 conjugation by the E2 SUMO-1 conjugating enzyme Ubc9. The Journal of biological chemistry 276, 40389-40395.

Cahilly-Snyder, L., Yang-Feng, T., Francke, U., and George, D.L. (1987). Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line. Somatic cell and molecular genetics 13, 235-244.

Carlsson, P., and Mahlapuu, M. (2002). Forkhead transcription factors: key players in development and metabolism. Developmental biology 250, 1-23.

Carter, M.E., and Brunet, A. (2007). FOXO transcription factors. Curr Biol 17, R113-114.

Caspari, T. (2000). How to activate p53. Curr Biol 10, R315-317.

Castrillon, D.H., Miao, L., Kollipara, R., Horner, J.W., and DePinho, R.A. (2003). Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301, 215-218.

- 142 - Catzavelos, C., Bhattacharya, N., Ung, Y.C., Wilson, J.A., Roncari, L., Sandhu, C., Shaw, P., Yeger, H., Morava-Protzner, I., Kapusta, L., Franssen, E., Pritchard, K.I., and Slingerland, J.M. (1997). Decreased levels of the cell-cycle inhibitor p27Kip1 protein: prognostic implications in primary breast cancer. Nature medicine 3, 227-230.

Chen, A., Mannen, H., and Li, S.S. (1998). Characterization of mouse ubiquitin-like SMT3A and SMT3B cDNAs and gene/. Biochemistry and molecular biology international 46, 1161-1174.

Chen, J., Lin, J., and Levine, A.J. (1995). Regulation of transcription functions of the p53 tumor suppressor by the mdm-2 oncogene. Molecular medicine (Cambridge, Mass 1, 142-152.

Chen, J., Marechal, V., and Levine, A.J. (1993). Mapping of the p53 and mdm-2 interaction domains. Molecular and cellular biology 13, 4107-4114.

Chen, J., Wu, X., Lin, J., and Levine, A.J. (1996). mdm-2 inhibits the G1 arrest and apoptosis functions of the p53 tumor suppressor protein. Molecular and cellular biology 16, 2445-2452.

Chen, J., Yusuf, I., Andersen, H.M., and Fruman, D.A. (2006). FOXO transcription factors cooperate with delta EF1 to activate growth suppressive genes in B lymphocytes. J Immunol 176, 2711-2721.

Chen, L., Marechal, V., Moreau, J., Levine, A.J., and Chen, J. (1997). Proteolytic cleavage of the mdm2 oncoprotein during apoptosis. The Journal of biological chemistry 272, 22966-22973.

Chevillard-Briet, M., Trouche, D., and Vandel, L. (2002). Control of CBP co-activating activity by arginine methylation. The EMBO journal 21, 5457-5466.

Chua, K.F., Mostoslavsky, R., Lombard, D.B., Pang, W.W., Saito, S., Franco, S., Kaushal, D., Cheng, H.L., Fischer, M.R., Stokes, N., Murphy, M.M., Appella, E., and Alt, F.W. (2005). Mammalian SIRT1 limits replicative life span in response to chronic genotoxic stress. Cell metabolism 2, 67-76.

Ciechanover, A., and Schwartz, A.L. (2002). Ubiquitin-mediated degradation of cellular proteins in health and disease. Hepatology (Baltimore, Md 35, 3-6.

Classon, M., Salama, S., Gorka, C., Mulloy, R., Braun, P., and Harlow, E. (2000). Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control. Proceedings of the National Academy of Sciences of the United States of America 97, 10820-10825.

- 143 - Cohen, T., and Yao, T.P. (2004). AcK-knowledge reversible acetylation. Sci STKE 2004, pe42.

Czech, M.P. (2003). Insulin's expanding control of forkheads. Proceedings of the National Academy of Sciences of the United States of America 100, 11198-11200.

D'Andrea, A., and Pellman, D. (1998). Deubiquitinating enzymes: a new class of biological regulators. Critical reviews in biochemistry and molecular biology 33, 337-352.

Dai, M.S., Zeng, S.X., Jin, Y., Sun, X.X., David, L., and Lu, H. (2004). Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition. Molecular and cellular biology 24, 7654-7668.

Daitoku, H., Hatta, M., Matsuzaki, H., Aratani, S., Ohshima, T., Miyagishi, M., Nakajima, T., and Fukamizu, A. (2004). Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity. Proceedings of the National Academy of Sciences of the United States of America 101, 10042-10047.

Dansen, T.B., Kops, G.J., Denis, S., Jelluma, N., Wanders, R.J., Bos, J.L., Burgering, B.M., and Wirtz, K.W. (2004). Regulation of sterol carrier protein gene expression by the forkhead transcription factor FOXO3a. Journal of lipid research 45, 81-88.

Daujat, S., Neel, H., and Piette, J. (2001). MDM2: life without p53. Trends Genet 17, 459-464.

Dazard, J.E., Augias, D., Neel, H., Mils, V., Marechal, V., Basset-Seguin, N., and Piette, J. (1997). MDM-2 protein is expressed in different layers of normal human skin. Oncogene 14, 1123-1128.

De Baere, E., Dixon, M.J., Small, K.W., Jabs, E.W., Leroy, B.P., Devriendt, K., Gillerot, Y., Mortier, G., Meire, F., Van Maldergem, L., Courtens, W., Hjalgrim, H., Huang, S., Liebaers, I., Van Regemorter, N., Touraine, P., Praphanphoj, V., Verloes, A., Udar, N., Yellore, V., Chalukya, M., Yelchits, S., De Paepe, A., Kuttenn, F., Fellous, M., Veitia, R., and Messiaen, L. (2001). Spectrum of FOXL2 gene mutations in blepharophimosis- ptosis-epicanthus inversus (BPES) families demonstrates a genotype--phenotype correlation. Human molecular genetics 10, 1591-1600.

De Baere, E., Lemercier, B., Christin-Maitre, S., Durval, D., Messiaen, L., Fellous, M., and Veitia, R. (2002). FOXL2 mutation screening in a large panel of POF patients and XX males. Journal of medical genetics 39, e43. de Ruijter, A.J., van Gennip, A.H., Caron, H.N., Kemp, S., and van Kuilenburg, A.B. (2003). Histone deacetylases (HDACs): characterization of the classical HDAC family. The Biochemical journal 370, 737-749.

- 144 - Deguchi, K., Ayton, P.M., Carapeti, M., Kutok, J.L., Snyder, C.S., Williams, I.R., Cross, N.C., Glass, C.K., Cleary, M.L., and Gilliland, D.G. (2003). MOZ-TIF2-induced acute myeloid leukemia requires the MOZ nucleosome binding motif and TIF2-mediated recruitment of CBP. Cancer cell 3, 259-271.

Desterro, J.M., Rodriguez, M.S., and Hay, R.T. (1998). SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Molecular cell 2, 233-239.

Epps, J.L., and Tanda, S. (1998). The Drosophila semushi mutation blocks nuclear import of bicoid during embryogenesis. Curr Biol 8, 1277-1280.

Essers, M.A., de Vries-Smits, L.M., Barker, N., Polderman, P.E., Burgering, B.M., and Korswagen, H.C. (2005). Functional interaction between beta-catenin and FOXO in oxidative stress signaling. Science 308, 1181-1184.

Essers, M.A., Weijzen, S., de Vries-Smits, A.M., Saarloos, I., de Ruiter, N.D., Bos, J.L., and Burgering, B.M. (2004). FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK. The EMBO journal 23, 4802-4812.

Fakharzadeh, S.S., Trusko, S.P., and George, D.L. (1991). Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line. The EMBO journal 10, 1565-1569.

Fang, S., Jensen, J.P., Ludwig, R.L., Vousden, K.H., and Weissman, A.M. (2000). Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. The Journal of biological chemistry 275, 8945-8951.

Feng, J., Tamaskovic, R., Yang, Z., Brazil, D.P., Merlo, A., Hess, D., and Hemmings, B.A. (2004). Stabilization of Mdm2 via decreased ubiquitination is mediated by protein kinase B/Akt-dependent phosphorylation. The Journal of biological chemistry 279, 35510-35517.

Fiddler, T.A., Smith, L., Tapscott, S.J., and Thayer, M.J. (1996). Amplification of MDM2 inhibits MyoD-mediated myogenesis. Molecular and cellular biology 16, 5048-5057.

Fornace, A.J., Jr., Nebert, D.W., Hollander, M.C., Luethy, J.D., Papathanasiou, M., Fargnoli, J., and Holbrook, N.J. (1989). Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. Molecular and cellular biology 9, 4196- 4203.

Frank, J., Pignata, C., Panteleyev, A.A., Prowse, D.M., Baden, H., Weiner, L., Gaetaniello, L., Ahmad, W., Pozzi, N., Cserhalmi-Friedman, P.B., Aita, V.M., Uyttendaele, H., Gordon, D., Ott, J., Brissette, J.L., and Christiano, A.M. (1999). Exposing the human nude phenotype. Nature 398, 473-474.

- 145 - Fraser, A.G., Kamath, R.S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., and Ahringer, J. (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325-330.

Freedman, D.A., and Levine, A.J. (1999). Regulation of the p53 protein by the MDM2 oncoprotein--thirty-eighth G.H.A. Clowes Memorial Award Lecture. Cancer Res 59, 1-7.

Fukuoka, M., Daitoku, H., Hatta, M., Matsuzaki, H., Umemura, S., and Fukamizu, A. (2003). Negative regulation of forkhead transcription factor AFX (Foxo4) by CBP- induced acetylation. International journal of molecular medicine 12, 503-508.

Furukawa-Hibi, Y., Yoshida-Araki, K., Ohta, T., Ikeda, K., and Motoyama, N. (2002). FOXO forkhead transcription factors induce G(2)-M checkpoint in response to oxidative stress. The Journal of biological chemistry 277, 26729-26732.

Furuyama, T., Kitayama, K., Yamashita, H., and Mori, N. (2003). Forkhead transcription factor FOXO1 (FKHR)-dependent induction of PDK4 gene expression in skeletal muscle during energy deprivation. The Biochemical journal 375, 365-371.

Galili, N., Davis, R.J., Fredericks, W.J., Mukhopadhyay, S., Rauscher, F.J., 3rd, Emanuel, B.S., Rovera, G., and Barr, F.G. (1993). Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nature genetics 5, 230-235.

Ganguli, G., and Wasylyk, B. (2003). p53-independent functions of MDM2. Mol Cancer Res 1, 1027-1035.

Gao, N., Zhang, J., Rao, M.A., Case, T.C., Mirosevich, J., Wang, Y., Jin, R., Gupta, A., Rennie, P.S., and Matusik, R.J. (2003). The role of hepatocyte nuclear factor-3 alpha (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes. Molecular endocrinology (Baltimore, Md 17, 1484-1507.

Gayther, S.A., Batley, S.J., Linger, L., Bannister, A., Thorpe, K., Chin, S.F., Daigo, Y., Russell, P., Wilson, A., Sowter, H.M., Delhanty, J.D., Ponder, B.A., Kouzarides, T., and Caldas, C. (2000). Mutations truncating the EP300 acetylase in human cancers. Nature genetics 24, 300-303.

Geyer, R.K., Yu, Z.K., and Maki, C.G. (2000). The MDM2 RING-finger domain is required to promote p53 nuclear export. Nature cell biology 2, 569-573.

Giannakou, M.E., Goss, M., Junger, M.A., Hafen, E., Leevers, S.J., and Partridge, L. (2004). Long-lived Drosophila with overexpressed dFOXO in adult fat body. Science 305, 361.

Giannakou, M.E., and Partridge, L. (2004). The interaction between FOXO and SIRT1: tipping the balance towards survival. Trends in cell biology 14, 408-412.

- 146 - Gillett, C.E., Smith, P., Peters, G., Lu, X., and Barnes, D.M. (1999). Cyclin-dependent kinase inhibitor p27Kip1 expression and interaction with other cell cycle-associated proteins in mammary carcinoma. The Journal of pathology 187, 200-206.

Gilley, J., Coffer, P.J., and Ham, J. (2003). FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. The Journal of cell biology 162, 613-622.

Girdwood, D., Bumpass, D., Vaughan, O.A., Thain, A., Anderson, L.A., Snowden, A.W., Garcia-Wilson, E., Perkins, N.D., and Hay, R.T. (2003). P300 transcriptional repression is mediated by SUMO modification. Molecular cell 11, 1043-1054.

Glauser, D.A., and Schlegel, W. (2007). The emerging role of FOXO transcription factors in pancreatic beta cells. The Journal of endocrinology 193, 195-207.

Goldberg, Z., Vogt Sionov, R., Berger, M., Zwang, Y., Perets, R., Van Etten, R.A., Oren, M., Taya, Y., and Haupt, Y. (2002). Tyrosine phosphorylation of Mdm2 by c-Abl: implications for p53 regulation. The EMBO journal 21, 3715-3727.

Gong, J.G., Costanzo, A., Yang, H.Q., Melino, G., Kaelin, W.G., Jr., Levrero, M., and Wang, J.Y. (1999). The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 399, 806-809.

Goodman, R.H., and Smolik, S. (2000). CBP/p300 in cell growth, transformation, and development. Genes & development 14, 1553-1577.

Gotz, C., Kartarius, S., Scholtes, P., Nastainczyk, W., and Montenarh, M. (1999). Identification of a CK2 phosphorylation site in mdm2. European journal of biochemistry / FEBS 266, 493-501.

Granadino, B., Arias-de-la-Fuente, C., Perez-Sanchez, C., Parraga, M., Lopez- Fernandez, L.A., del Mazo, J., and Rey-Campos, J. (2000). Fhx (Foxj2) expression is activated during spermatogenesis and very early in embryonic development. Mechanisms of development 97, 157-160.

Gray, D.A., Inazawa, J., Gupta, K., Wong, A., Ueda, R., and Takahashi, T. (1995). Elevated expression of Unph, a proto-oncogene at 3p21.3, in human lung tumors. Oncogene 10, 2179-2183.

Greer, E.L., and Brunet, A. (2005). FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24, 7410-7425.

Grossman, S.R., Perez, M., Kung, A.L., Joseph, M., Mansur, C., Xiao, Z.X., Kumar, S., Howley, P.M., and Livingston, D.M. (1998). p300/MDM2 complexes participate in MDM2-mediated p53 degradation. Molecular cell 2, 405-415.

- 147 - Guo, Y., Costa, R., Ramsey, H., Starnes, T., Vance, G., Robertson, K., Kelley, M., Reinbold, R., Scholer, H., and Hromas, R. (2002). The embryonic stem cell transcription factors Oct-4 and FoxD3 interact to regulate endodermal-specific promoter expression. Proceedings of the National Academy of Sciences of the United States of America 99, 3663-3667.

Gusterson, R.J., Jazrawi, E., Adcock, I.M., and Latchman, D.S. (2003). The transcriptional co-activators CREB-binding protein (CBP) and p300 play a critical role in cardiac hypertrophy that is dependent on their histone acetyltransferase activity. The Journal of biological chemistry 278, 6838-6847.

Hartwell, L.H., and Weinert, T.A. (1989). Checkpoints: controls that ensure the order of cell cycle events. Science 246, 629-634.

Hathaway, G.M., and Traugh, J.A. (1979). Cyclic nucleotide-independent protein kinases from rabbit reticulocytes. Purification of casein kinases. The Journal of biological chemistry 254, 762-768.

Hatini, V., Huh, S.O., Herzlinger, D., Soares, V.C., and Lai, E. (1996). Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2. Genes & development 10, 1467-1478.

Hauck, L., Harms, C., Grothe, D., An, J., Gertz, K., Kronenberg, G., Dietz, R., Endres, M., and von Harsdorf, R. (2007). Critical role for FoxO3a-dependent regulation of p21CIP1/WAF1 in response to statin signaling in cardiac myocytes. Circulation research 100, 50-60.

Haupt, Y., Maya, R., Kazaz, A., and Oren, M. (1997). Mdm2 promotes the rapid degradation of p53. Nature 387, 296-299.

Hawke, T.J., Jiang, N., and Garry, D.J. (2003). Absence of p21CIP rescues myogenic progenitor cell proliferative and regenerative capacity in Foxk1 null mice. The Journal of biological chemistry 278, 4015-4020.

Hay, T.J., and Meek, D.W. (2000). Multiple sites of in vivo phosphorylation in the MDM2 oncoprotein cluster within two important functional domains. FEBS letters 478, 183-186.

Hayashi, T., Seki, M., Maeda, D., Wang, W., Kawabe, Y., Seki, T., Saitoh, H., Fukagawa, T., Yagi, H., and Enomoto, T. (2002). Ubc9 is essential for viability of higher eukaryotic cells. Experimental cell research 280, 212-221.

Henning, W., Rohaly, G., Kolzau, T., Knippschild, U., Maacke, H., and Deppert, W. (1997). MDM2 is a target of simian virus 40 in cellular transformation and during lytic infection. Journal of virology 71, 7609-7618.

- 148 - Hershko, A., and Ciechanover, A. (1998). The ubiquitin system. Annual review of biochemistry 67, 425-479.

Hicke, L. (2001). Protein regulation by monoubiquitin. Nat Rev Mol Cell Biol 2, 195-201.

Hillion, J., Le Coniat, M., Jonveaux, P., Berger, R., and Bernard, O.A. (1997). AF6q21, a novel partner of the MLL gene in t(6;11)(q21;q23), defines a forkhead transcriptional factor subfamily. Blood 90, 3714-3719.

Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G., and Jentsch, S. (2002). RAD6- dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135-141.

Hoekman, M.F., Jacobs, F.M., Smidt, M.P., and Burbach, J.P. (2006). Spatial and temporal expression of FoxO transcription factors in the developing and adult murine brain. Gene Expr Patterns 6, 134-140.

Holowaty, M.N., Sheng, Y., Nguyen, T., Arrowsmith, C., and Frappier, L. (2003). Protein interaction domains of the ubiquitin-specific protease, USP7/HAUSP. The Journal of biological chemistry 278, 47753-47761.

Holzenberger, M., Dupont, J., Ducos, B., Leneuve, P., Geloen, A., Even, P.C., Cervera, P., and Le Bouc, Y. (2003). IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature 421, 182-187.

Honda, R., Tanaka, H., and Yasuda, H. (1997). Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS letters 420, 25-27.

Honda, R., and Yasuda, H. (1999). Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53. The EMBO journal 18, 22-27.

Honda, R., and Yasuda, H. (2000). Activity of MDM2, a ubiquitin ligase, toward p53 or itself is dependent on the RING finger domain of the ligase. Oncogene 19, 1473-1476.

Hong, H.K., Noveroske, J.K., Headon, D.J., Liu, T., Sy, M.S., Justice, M.J., and Chakravarti, A. (2001). The winged helix/forkhead transcription factor Foxq1 regulates differentiation of hair in satin mice. Genesis 29, 163-171.

Horne, M.C., Donaldson, K.L., Goolsby, G.L., Tran, D., Mulheisen, M., Hell, J.W., and Wahl, A.F. (1997). Cyclin G2 is up-regulated during growth inhibition and B cell antigen receptor-mediated cell cycle arrest. The Journal of biological chemistry 272, 12650- 12661.

Horne, M.C., Goolsby, G.L., Donaldson, K.L., Tran, D., Neubauer, M., and Wahl, A.F. (1996). Cyclin G1 and cyclin G2 comprise a new family of cyclins with contrasting tissue- - 149 - specific and cell cycle-regulated expression. The Journal of biological chemistry 271, 6050-6061.

Hosaka, T., Biggs, W.H., 3rd, Tieu, D., Boyer, A.D., Varki, N.M., Cavenee, W.K., and Arden, K.C. (2004). Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proceedings of the National Academy of Sciences of the United States of America 101, 2975-2980.

Howe, K., Williamson, J., Boddy, N., Sheer, D., Freemont, P., and Solomon, E. (1998). The ubiquitin-homology gene PIC1: characterization of mouse (Pic1) and human (UBL1) genes and pseudogenes. Genomics 47, 92-100.

Howitz, K.T., Bitterman, K.J., Cohen, H.Y., Lamming, D.W., Lavu, S., Wood, J.G., Zipkin, R.E., Chung, P., Kisielewski, A., Zhang, L.L., Scherer, B., and Sinclair, D.A. (2003). Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425, 191-196.

Hu, M., Li, P., Li, M., Li, W., Yao, T., Wu, J.W., Gu, W., Cohen, R.E., and Shi, Y. (2002). Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 111, 1041-1054.

Hu, M.C., Lee, D.F., Xia, W., Golfman, L.S., Ou-Yang, F., Yang, J.Y., Zou, Y., Bao, S., Hanada, N., Saso, H., Kobayashi, R., and Hung, M.C. (2004). IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 117, 225-237.

Huang, H., Regan, K.M., Lou, Z., Chen, J., and Tindall, D.J. (2006). CDK2-dependent phosphorylation of FOXO1 as an apoptotic response to DNA damage. Science 314, 294-297.

Huang, H., Regan, K.M., Wang, F., Wang, D., Smith, D.I., van Deursen, J.M., and Tindall, D.J. (2005). Skp2 inhibits FOXO1 in tumor suppression through ubiquitin- mediated degradation. Proceedings of the National Academy of Sciences of the United States of America 102, 1649-1654.

Huang, Y., Baker, R.T., and Fischer-Vize, J.A. (1995). Control of cell fate by a deubiquitinating enzyme encoded by the fat facets gene. Science 270, 1828-1831.

Huang, Y., and Fischer-Vize, J.A. (1996). Undifferentiated cells in the developing Drosophila eye influence facet assembly and require the Fat facets ubiquitin-specific protease. Development (Cambridge, England) 122, 3207-3216.

Hulander, M., Kiernan, A.E., Blomqvist, S.R., Carlsson, P., Samuelsson, E.J., Johansson, B.R., Steel, K.P., and Enerback, S. (2003). Lack of pendrin expression leads to deafness and expansion of the endolymphatic compartment in inner ears of Foxi1 null mutant mice. Development (Cambridge, England) 130, 2013-2025.

- 150 - Hurst, J.A., Baraitser, M., Auger, E., Graham, F., and Norell, S. (1990). An extended family with a dominantly inherited speech disorder. Developmental medicine and child neurology 32, 352-355.

Hwangbo, D.S., Gershman, B., Tu, M.P., Palmer, M., and Tatar, M. (2004). Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature 429, 562-566.

Imai, S., Johnson, F.B., Marciniak, R.A., McVey, M., Park, P.U., and Guarente, L. (2000). Sir2: an NAD-dependent that connects chromatin silencing, metabolism, and aging. Cold Spring Harbor symposia on quantitative biology 65, 297- 302.

Ito, A., Kawaguchi, Y., Lai, C.H., Kovacs, J.J., Higashimoto, Y., Appella, E., and Yao, T.P. (2002). MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation. The EMBO journal 21, 6236-6245.

Jacobs, F.M., van der Heide, L.P., Wijchers, P.J., Burbach, J.P., Hoekman, M.F., and Smidt, M.P. (2003). FoxO6, a novel member of the FoxO class of transcription factors with distinct shuttling dynamics. The Journal of biological chemistry 278, 35959-35967.

Jensen, D.E., Proctor, M., Marquis, S.T., Gardner, H.P., Ha, S.I., Chodosh, L.A., Ishov, A.M., Tommerup, N., Vissing, H., Sekido, Y., Minna, J., Borodovsky, A., Schultz, D.C., Wilkinson, K.D., Maul, G.G., Barlev, N., Berger, S.L., Prendergast, G.C., and Rauscher, F.J., 3rd (1998). BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression. Oncogene 16, 1097- 1112.

Jeong, J., Juhn, K., Lee, H., Kim, S.H., Min, B.H., Lee, K.M., Cho, M.H., Park, G.H., and Lee, K.H. (2007). SIRT1 promotes DNA repair activity and deacetylation of Ku70. Experimental & molecular medicine 39, 8-13.

Jeong, J.W., Bae, M.K., Ahn, M.Y., Kim, S.H., Sohn, T.K., Bae, M.H., Yoo, M.A., Song, E.J., Lee, K.J., and Kim, K.W. (2002). Regulation and destabilization of HIF-1alpha by ARD1-mediated acetylation. Cell 111, 709-720.

Jiang, M., Shao, Z.M., Wu, J., Lu, J.S., Yu, L.M., Yuan, J.D., Han, Q.X., Shen, Z.Z., and Fontana, J.A. (1997). p21/waf1/cip1 and mdm-2 expression in breast carcinoma patients as related to prognosis. International journal of cancer 74, 529-534.

Jin, S., and Levine, A.J. (2001). The p53 functional circuit. Journal of cell science 114, 4139-4140.

Joanisse, D.R., Inaguma, Y., and Tanguay, R.M. (1998). Cloning and developmental expression of a nuclear ubiquitin-conjugating enzyme (DmUbc9) that interacts with small

- 151 - heat shock proteins in Drosophila melanogaster. Biochemical and biophysical research communications 244, 102-109.

Johnson, E.S. (2004). Protein modification by SUMO. Annual review of biochemistry 73, 355-382.

Jones, D., Crowe, E., Stevens, T.A., and Candido, E.P. (2002). Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin- conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins. Genome biology 3, RESEARCH0002.

Jones, S.N., Roe, A.E., Donehower, L.A., and Bradley, A. (1995). Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208.

Junger, M.A., Rintelen, F., Stocker, H., Wasserman, J.D., Vegh, M., Radimerski, T., Greenberg, M.E., and Hafen, E. (2003). The Drosophila forkhead transcription factor FOXO mediates the reduction in cell number associated with reduced insulin signaling. Journal of biology 2, 20.

Juven-Gershon, T., Shifman, O., Unger, T., Elkeles, A., Haupt, Y., and Oren, M. (1998). The Mdm2 oncoprotein interacts with the cell fate regulator Numb. Molecular and cellular biology 18, 3974-3982.

Kadoya, T., Yamamoto, H., Suzuki, T., Yukita, A., Fukui, A., Michiue, T., Asahara, T., Tanaka, K., Asashima, M., and Kikuchi, A. (2002). Desumoylation activity of Axam, a novel Axin-binding protein, is involved in downregulation of beta-catenin. Molecular and cellular biology 22, 3803-3819.

Kaeberlein, M., McVey, M., and Guarente, L. (1999). The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes & development 13, 2570-2580.

Kaestner, K.H. (2000). The hepatocyte nuclear factor 3 (HNF3 or FOXA) family in metabolism. Trends in endocrinology and metabolism: TEM 11, 281-285.

Kaestner, K.H., Hiemisch, H., Luckow, B., and Schutz, G. (1994). The HNF-3 gene family of transcription factors in mice: gene structure, cDNA sequence, and mRNA distribution. Genomics 20, 377-385.

Kaestner, K.H., Knochel, W., and Martinez, D.E. (2000). Unified nomenclature for the winged helix/forkhead transcription factors. Genes & development 14, 142-146.

Kaestner, K.H., Silberg, D.G., Traber, P.G., and Schutz, G. (1997). The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation. Genes & development 11, 1583-1595.

- 152 - Kajihara, T., Jones, M., Fusi, L., Takano, M., Feroze-Zaidi, F., Pirianov, G., Mehmet, H., Ishihara, O., Higham, J.M., Lam, E.W., and Brosens, J.J. (2006). Differential expression of FOXO1 and FOXO3a confers resistance to oxidative cell death upon endometrial decidualization. Molecular endocrinology (Baltimore, Md 20, 2444-2455.

Kalinichenko, V.V., Lim, L., Stolz, D.B., Shin, B., Rausa, F.M., Clark, J., Whitsett, J.A., Watkins, S.C., and Costa, R.H. (2001). Defects in pulmonary vasculature and perinatal lung hemorrhage in mice heterozygous null for the Forkhead Box f1 transcription factor. Developmental biology 235, 489-506.

Kalinichenko, V.V., Zhou, Y., Shin, B., Stolz, D.B., Watkins, S.C., Whitsett, J.A., and Costa, R.H. (2002). Wild-type levels of the mouse Forkhead Box f1 gene are essential for lung repair. American journal of physiology 282, L1253-1265.

Kamei, Y., Mizukami, J., Miura, S., Suzuki, M., Takahashi, N., Kawada, T., Taniguchi, T., and Ezaki, O. (2003). A forkhead transcription factor FKHR up-regulates lipoprotein lipase expression in skeletal muscle. FEBS letters 536, 232-236.

Kamitani, T., Kito, K., Nguyen, H.P., Fukuda-Kamitani, T., and Yeh, E.T. (1998). Characterization of a second member of the sentrin family of ubiquitin-like proteins. The Journal of biological chemistry 273, 11349-11353.

Katoh, M., and Katoh, M. (2004a). Germ-line mutation of Foxn5 gene in mouse lineage. International journal of molecular medicine 14, 463-467.

Katoh, M., and Katoh, M. (2004b). Human FOX gene family (Review). International journal of oncology 25, 1495-1500.

Katoh, M., and Katoh, M. (2004c). Identification and characterization of human FOXN5 and rat Foxn5 genes in silico. International journal of oncology 24, 1339-1344.

Katoh, M., and Katoh, M. (2004d). Identification and characterization of human FOXN6, mouse Foxn6, and rat Foxn6 genes in silico. International journal of oncology 25, 219- 223.

Kenyon, C., Chang, J., Gensch, E., Rudner, A., and Tabtiang, R. (1993). A C. elegans mutant that lives twice as long as wild type. Nature 366, 461-464.

Kim, K.I., Baek, S.H., Jeon, Y.J., Nishimori, S., Suzuki, T., Uchida, S., Shimbara, N., Saitoh, H., Tanaka, K., and Chung, C.H. (2000). A new SUMO-1-specific protease, SUSP1, that is highly expressed in reproductive organs. The Journal of biological chemistry 275, 14102-14106.

- 153 - Kimura, K.D., Tissenbaum, H.A., Liu, Y., and Ruvkun, G. (1997). daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277, 942-946.

Kirkwood, T.B., and Austad, S.N. (2000). Why do we age? Nature 408, 233-238.

Kitamura, T., Nakae, J., Kitamura, Y., Kido, Y., Biggs, W.H., 3rd, Wright, C.V., White, M.F., Arden, K.C., and Accili, D. (2002). The forkhead transcription factor Foxo1 links insulin signaling to Pdx1 regulation of pancreatic beta cell growth. The Journal of clinical investigation 110, 1839-1847.

Kitamura, Y.I., Kitamura, T., Kruse, J.P., Raum, J.C., Stein, R., Gu, W., and Accili, D. (2005). FoxO1 protects against pancreatic beta cell failure through NeuroD and MafA induction. Cell metabolism 2, 153-163.

Kloetzli, J.M., Fontaine-Glover, I.A., Brown, E.R., Kuo, M., and Labosky, P.A. (2001). The winged helix gene, Foxb1, controls development of mammary glands and regions of the CNS that regulate the milk-ejection reflex. Genesis 29, 60-71.

Kobayashi, T., and Cohen, P. (1999). Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3- phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. The Biochemical journal 339 ( Pt 2), 319-328.

Kops, G.J., Dansen, T.B., Polderman, P.E., Saarloos, I., Wirtz, K.W., Coffer, P.J., Huang, T.T., Bos, J.L., Medema, R.H., and Burgering, B.M. (2002). Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316-321.

Korver, W., Roose, J., and Clevers, H. (1997). The winged-helix transcription factor Trident is expressed in cycling cells. Nucleic acids research 25, 1715-1719.

Kramer, J.M., Davidge, J.T., Lockyer, J.M., and Staveley, B.E. (2003). Expression of Drosophila FOXO regulates growth and can phenocopy starvation. BMC developmental biology 3, 5.

Kubbutat, M.H., Jones, S.N., and Vousden, K.H. (1997). Regulation of p53 stability by Mdm2. Nature 387, 299-303.

Kume, T., Deng, K., and Hogan, B.L. (2000). Murine forkhead/winged helix genes Foxc1 (Mf1) and Foxc2 (Mfh1) are required for the early organogenesis of the kidney and urinary tract. Development (Cambridge, England) 127, 1387-1395.

- 154 - Kume, T., Deng, K.Y., Winfrey, V., Gould, D.B., Walter, M.A., and Hogan, B.L. (1998). The forkhead/winged helix gene Mf1 is disrupted in the pleiotropic mouse mutation congenital hydrocephalus. Cell 93, 985-996.

Kurepa, J., Walker, J.M., Smalle, J., Gosink, M.M., Davis, S.J., Durham, T.L., Sung, D.Y., and Vierstra, R.D. (2003). The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and -2 conjugates is increased by stress. The Journal of biological chemistry 278, 6862-6872.

Kussie, P.H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A.J., and Pavletich, N.P. (1996). Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 274, 948-953.

Kwok, R.P., Liu, X.T., and Smith, G.D. (2006). Distribution of co-activators CBP and p300 during mouse oocyte and embryo development. Molecular reproduction and development 73, 885-894.

Kwon, H.S., Huang, B., Unterman, T.G., and Harris, R.A. (2004). Protein kinase B-alpha inhibits human pyruvate dehydrogenase kinase-4 gene induction by dexamethasone through inactivation of FOXO transcription factors. Diabetes 53, 899-910.

Kyoung Kim, H., Kyoung Kim, Y., Song, I.H., Baek, S.H., Lee, S.R., Hye Kim, J., and Kim, J.R. (2005). Down-regulation of a forkhead transcription factor, FOXO3a, accelerates cellular senescence in human dermal fibroblasts. J Gerontol A Biol Sci Med Sci 60, 4-9.

Lai, C.S., Fisher, S.E., Hurst, J.A., Vargha-Khadem, F., and Monaco, A.P. (2001). A forkhead-domain gene is mutated in a severe speech and language disorder. Nature 413, 519-523.

Lam, E.W., Francis, R.E., and Petkovic, M. (2006). FOXO transcription factors: key regulators of cell fate. Biochemical Society transactions 34, 722-726.

Landgren, H., and Carlsson, P. (2004). FoxJ3, a novel mammalian forkhead gene expressed in neuroectoderm, neural crest, and myotome. Dev Dyn 231, 396-401.

Langley, E., Pearson, M., Faretta, M., Bauer, U.M., Frye, R.A., Minucci, S., Pelicci, P.G., and Kouzarides, T. (2002). Human SIR2 deacetylates p53 and antagonizes PML/p53- induced cellular senescence. The EMBO journal 21, 2383-2396.

Lawlor, M.A., and Rotwein, P. (2000). Insulin-like growth factor-mediated muscle cell survival: central roles for Akt and cyclin-dependent kinase inhibitor p21. Molecular and cellular biology 20, 8983-8995.

- 155 - Lee, P.S., Chang, C., Liu, D., and Derynck, R. (2003). Sumoylation of Smad4, the common Smad mediator of transforming growth factor-beta family signaling. The Journal of biological chemistry 278, 27853-27863.

Lee, T.A., and Tyers, M. (2001). Ubiquitin junction, what's your function? Genome biology 2, REPORTS4025.

Lehmann, O.J., Sowden, J.C., Carlsson, P., Jordan, T., and Bhattacharya, S.S. (2003). Fox's in development and disease. Trends Genet 19, 339-344.

Lehtinen, M.K., Yuan, Z., Boag, P.R., Yang, Y., Villen, J., Becker, E.B., DiBacco, S., de la Iglesia, N., Gygi, S., Blackwell, T.K., and Bonni, A. (2006). A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span. Cell 125, 987-1001.

Leibiger, I.B., and Berggren, P.O. (2006). Sirt1: a metabolic master switch that modulates lifespan. Nature medicine 12, 34-36; discussion 36.

Lengner, C.J., Steinman, H.A., Gagnon, J., Smith, T.W., Henderson, J.E., Kream, B.E., Stein, G.S., Lian, J.B., and Jones, S.N. (2006). Osteoblast differentiation and skeletal development are regulated by Mdm2-p53 signaling. The Journal of cell biology 172, 909- 921.

Li, C., Lusis, A.J., Sparkes, R., Tran, S.M., and Gaynor, R. (1992). Characterization and chromosomal mapping of the gene encoding the cellular DNA binding protein HTLF. Genomics 13, 658-664.

Li, M., Brooks, C.L., Wu-Baer, F., Chen, D., Baer, R., and Gu, W. (2003a). Mono- versus polyubiquitination: differential control of p53 fate by Mdm2. Science 302, 1972-1975.

Li, M., Luo, J., Brooks, C.L., and Gu, W. (2002). Acetylation of p53 inhibits its ubiquitination by Mdm2. The Journal of biological chemistry 277, 50607-50611.

Li, M., Zhang, Z., Hill, D.L., Chen, X., Wang, H., and Zhang, R. (2005). Genistein, a dietary isoflavone, down-regulates the MDM2 oncogene at both transcriptional and posttranslational levels. Cancer research 65, 8200-8208.

Li, P., Lee, H., Guo, S., Unterman, T.G., Jenster, G., and Bai, W. (2003b). AKT- independent protection of prostate cancer cells from apoptosis mediated through complex formation between the androgen receptor and FKHR. Molecular and cellular biology 23, 104-118.

Li, P., Nicosia, S.V., and Bai, W. (2001). Antagonism between PTEN/MMAC1/TEP-1 and androgen receptor in growth and apoptosis of prostatic cancer cells. The Journal of biological chemistry 276, 20444-20450.

- 156 - Li, S., Mo, Z., Yang, X., Price, S.M., Shen, M.M., and Xiang, M. (2004a). Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors. Neuron 43, 795-807.

Li, S., Zhou, D., Lu, M.M., and Morrisey, E.E. (2004b). Advanced cardiac morphogenesis does not require heart tube fusion. Science 305, 1619-1622.

Lin, H.K., Wang, L., Hu, Y.C., Altuwaijri, S., and Chang, C. (2002a). Phosphorylation- dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase. The EMBO journal 21, 4037-4048.

Lin, J., Chen, J., Elenbaas, B., and Levine, A.J. (1994). Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein. Genes & development 8, 1235-1246.

Lin, K., Hsin, H., Libina, N., and Kenyon, C. (2001). Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nature genetics 28, 139-145.

Lin, L., Miller, C.T., Contreras, J.I., Prescott, M.S., Dagenais, S.L., Wu, R., Yee, J., Orringer, M.B., Misek, D.E., Hanash, S.M., Glover, T.W., and Beer, D.G. (2002b). The hepatocyte nuclear factor 3 alpha gene, HNF3alpha (FOXA1), on chromosome band 14q13 is amplified and overexpressed in esophageal and lung adenocarcinomas. Cancer research 62, 5273-5279.

Lin, X., Liang, M., Liang, Y.Y., Brunicardi, F.C., and Feng, X.H. (2003a). SUMO-1/Ubc9 promotes nuclear accumulation and metabolic stability of tumor suppressor Smad4. The Journal of biological chemistry 278, 31043-31048.

Lin, X., Liang, M., Liang, Y.Y., Brunicardi, F.C., Melchior, F., and Feng, X.H. (2003b). Activation of transforming growth factor-beta signaling by SUMO-1 modification of tumor suppressor Smad4/DPC4. The Journal of biological chemistry 278, 18714-18719.

Lohrum, M.A., Ashcroft, M., Kubbutat, M.H., and Vousden, K.H. (2000). Identification of a cryptic nucleolar-localization signal in MDM2. Nature cell biology 2, 179-181.

Lohrum, M.A., Ludwig, R.L., Kubbutat, M.H., Hanlon, M., and Vousden, K.H. (2003). Regulation of HDM2 activity by the ribosomal protein L11. Cancer cell 3, 577-587.

Lois, L.M., Lima, C.D., and Chua, N.H. (2003). Small ubiquitin-like modifier modulates abscisic acid signaling in Arabidopsis. The Plant cell 15, 1347-1359.

Lundgren, K., Montes de Oca Luna, R., McNeill, Y.B., Emerick, E.P., Spencer, B., Barfield, C.R., Lozano, G., Rosenberg, M.P., and Finlay, C.A. (1997). Targeted

- 157 - expression of MDM2 uncouples S phase from mitosis and inhibits mammary gland development independent of p53. Genes & development 11, 714-725.

Luo, J., Nikolaev, A.Y., Imai, S., Chen, D., Su, F., Shiloh, A., Guarente, L., and Gu, W. (2001). Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell 107, 137-148.

Macri, E., and Loda, M. (1998). Role of p27 in prostate carcinogenesis. Cancer metastasis reviews 17, 337-344.

Mahmud, D.L., M, G.A., Deb, D.K., Platanias, L.C., Uddin, S., and Wickrema, A. (2002). Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells. Oncogene 21, 1556-1562.

Mannen, H., Tseng, H.M., Cho, C.L., and Li, S.S. (1996). Cloning and expression of human homolog HSMT3 to yeast SMT3 suppressor of MIF2 mutations in a centromere protein gene. Biochemical and biophysical research communications 222, 178-180.

Marchenko, N.D., Wolff, S., Erster, S., Becker, K., and Moll, U.M. (2007). Monoubiquitylation promotes mitochondrial p53 translocation. The EMBO journal 26, 923-934.

Marechal, V., Elenbaas, B., Piette, J., Nicolas, J.C., and Levine, A.J. (1994). The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes. Molecular and cellular biology 14, 7414-7420.

Martinez-Gac, L., Marques, M., Garcia, Z., Campanero, M.R., and Carrera, A.C. (2004). Control of cyclin G2 mRNA expression by forkhead transcription factors: novel mechanism for cell cycle control by phosphoinositide 3-kinase and forkhead. Molecular and cellular biology 24, 2181-2189.

Martinez, S.C., Cras-Meneur, C., Bernal-Mizrachi, E., and Permutt, M.A. (2006). Glucose regulates Foxo1 through insulin receptor signaling in the pancreatic islet beta- cell. Diabetes 55, 1581-1591.

Matsuzaki, H., Daitoku, H., Hatta, M., Aoyama, H., Yoshimochi, K., and Fukamizu, A. (2005). Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation. Proceedings of the National Academy of Sciences of the United States of America 102, 11278-11283.

Matsuzaki, H., Daitoku, H., Hatta, M., Tanaka, K., and Fukamizu, A. (2003). Insulin- induced phosphorylation of FKHR (Foxo1) targets to proteasomal degradation. Proceedings of the National Academy of Sciences of the United States of America 100, 11285-11290.

- 158 - Maya, R., Balass, M., Kim, S.T., Shkedy, D., Leal, J.F., Shifman, O., Moas, M., Buschmann, T., Ronai, Z., Shiloh, Y., Kastan, M.B., Katzir, E., and Oren, M. (2001). ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes & development 15, 1067-1077.

Mayo, L.D., and Donner, D.B. (2001). A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proceedings of the National Academy of Sciences of the United States of America 98, 11598-11603.

Mayo, L.D., Turchi, J.J., and Berberich, S.J. (1997). Mdm-2 phosphorylation by DNA- dependent protein kinase prevents interaction with p53. Cancer research 57, 5013-5016.

Mazet, F., Yu, J.K., Liberles, D.A., Holland, L.Z., and Shimeld, S.M. (2003). Phylogenetic relationships of the Fox (Forkhead) gene family in the Bilateria. Gene 316, 79-89.

McBurney, M.W., Yang, X., Jardine, K., Bieman, M., Th'ng, J., and Lemieux, M. (2003). The absence of SIR2alpha protein has no effect on global gene silencing in mouse embryonic stem cells. Mol Cancer Res 1, 402-409.

McCoy, M.A., Gesell, J.J., Senior, M.M., and Wyss, D.F. (2003). Flexible lid to the p53- binding domain of human Mdm2: implications for p53 regulation. Proceedings of the National Academy of Sciences of the United States of America 100, 1645-1648.

Medema, R.H., Kops, G.J., Bos, J.L., and Burgering, B.M. (2000). AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404, 782-787.

Meek, D.W. (1994). Post-translational modification of p53. Seminars in cancer biology 5, 203-210.

Mihara, M., Erster, S., Zaika, A., Petrenko, O., Chittenden, T., Pancoska, P., and Moll, U.M. (2003). p53 has a direct apoptogenic role at the mitochondria. Molecular cell 11, 577-590.

Mimnaugh, E.G., Chen, H.Y., Davie, J.R., Celis, J.E., and Neckers, L. (1997). Rapid deubiquitination of nucleosomal histones in human tumor cells caused by proteasome inhibitors and stress response inducers: effects on replication, transcription, translation, and the cellular stress response. Biochemistry 36, 14418-14429.

Minoretti, P., Arra, M., Emanuele, E., Olivieri, V., Aldeghi, A., Politi, P., Martinelli, V., Pesenti, S., and Falcone, C. (2007). A W148R mutation in the human FOXD4 gene segregating with dilated cardiomyopathy, obsessive-compulsive disorder, and suicidality. International journal of molecular medicine 19, 369-372.

- 159 - Miwa, S., Uchida, C., Kitagawa, K., Hattori, T., Oda, T., Sugimura, H., Yasuda, H., Nakamura, H., Chida, K., and Kitagawa, M. (2006). Mdm2-mediated pRB downregulation is involved in carcinogenesis in a p53-independent manner. Biochemical and biophysical research communications 340, 54-61.

Miyauchi, H., Minamino, T., Tateno, K., Kunieda, T., Toko, H., and Komuro, I. (2004). Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21- dependent pathway. The EMBO journal 23, 212-220.

Moazed, D., and Johnson, D. (1996). A deubiquitinating enzyme interacts with SIR4 and regulates silencing in S. cerevisiae. Cell 86, 667-677.

Modur, V., Nagarajan, R., Evers, B.M., and Milbrandt, J. (2002). FOXO proteins regulate tumor necrosis factor-related apoptosis inducing ligand expression. Implications for PTEN mutation in prostate cancer. The Journal of biological chemistry 277, 47928- 47937.

Momand, J., Zambetti, G.P., Olson, D.C., George, D., and Levine, A.J. (1992). The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53- mediated transactivation. Cell 69, 1237-1245.

Montes de Oca Luna, R., Wagner, D.S., and Lozano, G. (1995). Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206.

Mossessova, E., and Lima, C.D. (2000). Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Molecular cell 5, 865-876.

Motta, M.C., Divecha, N., Lemieux, M., Kamel, C., Chen, D., Gu, W., Bultsma, Y., McBurney, M., and Guarente, L. (2004). Mammalian SIRT1 represses forkhead transcription factors. Cell 116, 551-563.

Muraoka, M., Konishi, M., Kikuchi-Yanoshita, R., Tanaka, K., Shitara, N., Chong, J.M., Iwama, T., and Miyaki, M. (1996). p300 gene alterations in colorectal and gastric carcinomas. Oncogene 12, 1565-1569.

Nadal, A., Marrero, P.F., and Haro, D. (2002). Down-regulation of the mitochondrial 3- hydroxy-3-methylglutaryl-CoA synthase gene by insulin: the role of the forkhead transcription factor FKHRL1. The Biochemical journal 366, 289-297.

Nakae, J., Biggs, W.H., 3rd, Kitamura, T., Cavenee, W.K., Wright, C.V., Arden, K.C., and Accili, D. (2002). Regulation of insulin action and pancreatic beta-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1. Nature genetics 32, 245-253.

- 160 - Nakae, J., Cao, Y., Daitoku, H., Fukamizu, A., Ogawa, W., Yano, Y., and Hayashi, Y. (2006). The LXXLL motif of murine forkhead transcription factor FoxO1 mediates Sirt1- dependent transcriptional activity. The Journal of clinical investigation 116, 2473-2483.

Nakae, J., Kitamura, T., Kitamura, Y., Biggs, W.H., 3rd, Arden, K.C., and Accili, D. (2003). The forkhead transcription factor Foxo1 regulates adipocyte differentiation. Developmental cell 4, 119-129.

Nakae, J., Kitamura, T., Silver, D.L., and Accili, D. (2001). The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression. The Journal of clinical investigation 108, 1359-1367.

Nakamura, N., Ramaswamy, S., Vazquez, F., Signoretti, S., Loda, M., and Sellers, W.R. (2000). Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN. Molecular and cellular biology 20, 8969-8982.

Nakamura, T., Furukawa, Y., Nakagawa, H., Tsunoda, T., Ohigashi, H., Murata, K., Ishikawa, O., Ohgaki, K., Kashimura, N., Miyamoto, M., Hirano, S., Kondo, S., Katoh, H., Nakamura, Y., and Katagiri, T. (2004). Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection. Oncogene 23, 2385- 2400.

Ng, I.O., Lam, K.Y., Ng, M., and Regezi, J.A. (1999). Expression of p21/waf1 in oral squamous cell carcinomas--correlation with p53 and mdm2 and cellular proliferation index. Oral oncology 35, 63-69.

Nishida, T., Kaneko, F., Kitagawa, M., and Yasuda, H. (2001). Characterization of a novel mammalian SUMO-1/Smt3-specific , a homologue of rat axam, which is an axin-binding protein promoting beta-catenin degradation. The Journal of biological chemistry 276, 39060-39066.

Nishida, T., Tanaka, H., and Yasuda, H. (2000). A novel mammalian Smt3-specific isopeptidase 1 (SMT3IP1) localized in the nucleolus at interphase. European journal of biochemistry / FEBS 267, 6423-6427.

O'Connor, L., Strasser, A., O'Reilly, L.A., Hausmann, G., Adams, J.M., Cory, S., and Huang, D.C. (1998). Bim: a novel member of the Bcl-2 family that promotes apoptosis. The EMBO journal 17, 384-395.

Ogg, S., Paradis, S., Gottlieb, S., Patterson, G.I., Lee, L., Tissenbaum, H.A., and Ruvkun, G. (1997). The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994-999.

- 161 - Ogryzko, V.V., Schiltz, R.L., Russanova, V., Howard, B.H., and Nakatani, Y. (1996). The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953- 959.

Oliner, J.D., Kinzler, K.W., Meltzer, P.S., George, D.L., and Vogelstein, B. (1992). Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature 358, 80-83.

Olson, D.C., Marechal, V., Momand, J., Chen, J., Romocki, C., and Levine, A.J. (1993). Identification and characterization of multiple mdm-2 proteins and mdm-2-p53 protein complexes. Oncogene 8, 2353-2360.

Onuma, H., Vander Kooi, B.T., Boustead, J.N., Oeser, J.K., and O'Brien, R.M. (2006). Correlation between FOXO1a (FKHR) and FOXO3a (FKHRL1) binding and the inhibition of basal glucose-6-phosphatase catalytic subunit gene transcription by insulin. Molecular endocrinology (Baltimore, Md 20, 2831-2847.

Ortiz, L., Zannini, M., Di Lauro, R., and Santisteban, P. (1997). Transcriptional control of the forkhead thyroid transcription factor TTF-2 by thyrotropin, insulin, and insulin-like growth factor I. The Journal of biological chemistry 272, 23334-23339.

Pan, Y., and Chen, J. (2003). MDM2 promotes ubiquitination and degradation of MDMX. Molecular and cellular biology 23, 5113-5121.

Papa, F.R., and Hochstrasser, M. (1993). The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene. Nature 366, 313-319.

Papathanasiou, M.A., Kerr, N.C., Robbins, J.H., McBride, O.W., Alamo, I., Jr., Barrett, S.F., Hickson, I.D., and Fornace, A.J., Jr. (1991). Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C. Molecular and cellular biology 11, 1009-1016.

Parry, P., Wei, Y., and Evans, G. (1994). Cloning and characterization of the t(X;11) breakpoint from a leukemic cell line identify a new member of the forkhead gene family. Genes, & cancer 11, 79-84.

Pauley, S., Lai, E., and Fritzsch, B. (2006). Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear. Dev Dyn 235, 2470-2482.

Pelletier, G.J., Brody, S.L., Liapis, H., White, R.A., and Hackett, B.P. (1998). A human forkhead/winged-helix transcription factor expressed in developing pulmonary and renal epithelium. The American journal of physiology 274, L351-359.

- 162 - Peng, Y., Chen, L., Li, C., Lu, W., and Chen, J. (2001). Inhibition of MDM2 by contributes to mutant p53 stabilization. The Journal of biological chemistry 276, 40583- 40590.

Perrot, V., and Rechler, M.M. (2005). The coactivator p300 directly acetylates the forkhead transcription factor Foxo1 and stimulates Foxo1-induced transcription. Molecular endocrinology (Baltimore, Md 19, 2283-2298.

Perry, M.E., Piette, J., Zawadzki, J.A., Harvey, D., and Levine, A.J. (1993). The mdm-2 gene is induced in response to UV light in a p53-dependent manner. Proceedings of the National Academy of Sciences of the United States of America 90, 11623-11627.

Picard, F., Kurtev, M., Chung, N., Topark-Ngarm, A., Senawong, T., Machado De Oliveira, R., Leid, M., McBurney, M.W., and Guarente, L. (2004). Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma. Nature 429, 771-776.

Pickart, C.M. (2001). Mechanisms underlying ubiquitination. Annual review of biochemistry 70, 503-533.

Picksley, S.M., Vojtesek, B., Sparks, A., and Lane, D.P. (1994). Immunochemical analysis of the interaction of p53 with MDM2;--fine mapping of the MDM2 binding site on p53 using synthetic peptides. Oncogene 9, 2523-2529.

Plas, D.R., and Thompson, C.B. (2003). Akt activation promotes degradation of tuberin and FOXO3a via the proteasome. The Journal of biological chemistry 278, 12361- 12366.

Pochampally, R., Fodera, B., Chen, L., Lu, W., and Chen, J. (1999). Activation of an MDM2-specific caspase by p53 in the absence of apoptosis. The Journal of biological chemistry 274, 15271-15277.

Polesskaya, A., Duquet, A., Naguibneva, I., Weise, C., Vervisch, A., Bengal, E., Hucho, F., Robin, P., and Harel-Bellan, A. (2000). CREB-binding protein/p300 activates MyoD by acetylation. The Journal of biological chemistry 275, 34359-34364.

Potente, M., Urbich, C., Sasaki, K., Hofmann, W.K., Heeschen, C., Aicher, A., Kollipara, R., DePinho, R.A., Zeiher, A.M., and Dimmeler, S. (2005). Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization. The Journal of clinical investigation 115, 2382-2392.

Puig, O., Marr, M.T., Ruhf, M.L., and Tjian, R. (2003). Control of cell number by Drosophila FOXO: downstream and feedback regulation of the insulin receptor pathway. Genes & development 17, 2006-2020.

- 163 - Ramaswamy, S., Nakamura, N., Sansal, I., Bergeron, L., and Sellers, W.R. (2002). A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR. Cancer cell 2, 81-91.

Rena, G., Woods, Y.L., Prescott, A.R., Peggie, M., Unterman, T.G., Williams, M.R., and Cohen, P. (2002). Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion. The EMBO journal 21, 2263-2271.

Robzyk, K., Recht, J., and Osley, M.A. (2000). Rad6-dependent ubiquitination of histone H2B in yeast. Science 287, 501-504.

Rogina, B., and Helfand, S.L. (2004). Sir2 mediates longevity in the fly through a pathway related to calorie restriction. Proceedings of the National Academy of Sciences of the United States of America 101, 15998-16003.

Ross, S., Best, J.L., Zon, L.I., and Gill, G. (2002). SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization. Molecular cell 10, 831-842.

Sakaguchi, K., Herrera, J.E., Saito, S., Miki, T., Bustin, M., Vassilev, A., Anderson, C.W., and Appella, E. (1998). DNA damage activates p53 through a phosphorylation- acetylation cascade. Genes & development 12, 2831-2841.

Samuel, V.T., Choi, C.S., Phillips, T.G., Romanelli, A.J., Geisler, J.G., Bhanot, S., McKay, R., Monia, B., Shutter, J.R., Lindberg, R.A., Shulman, G.I., and Veniant, M.M. (2006). Targeting foxo1 in mice using antisense oligonucleotide improves hepatic and peripheral insulin action. Diabetes 55, 2042-2050.

Sandri, M., Sandri, C., Gilbert, A., Skurk, C., Calabria, E., Picard, A., Walsh, K., Schiaffino, S., Lecker, S.H., and Goldberg, A.L. (2004). Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117, 399-412.

Saucedo, L.J., Myers, C.D., and Perry, M.E. (1999). Multiple murine double minute gene 2 (MDM2) proteins are induced by ultraviolet light. The Journal of biological chemistry 274, 8161-8168.

Schmidt, M., Fernandez de Mattos, S., van der Horst, A., Klompmaker, R., Kops, G.J., Lam, E.W., Burgering, B.M., and Medema, R.H. (2002). Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D. Molecular and cellular biology 22, 7842-7852.

Scott, K.L., and Plon, S.E. (2005). CHES1/FOXN3 interacts with Ski-interacting protein and acts as a transcriptional repressor. Gene 359, 119-126.

- 164 - Seoane, J., Le, H.V., Shen, L., Anderson, S.A., and Massague, J. (2004). Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 117, 211-223.

Shen, Z., Pardington-Purtymun, P.E., Comeaux, J.C., Moyzis, R.K., and Chen, D.J. (1996). UBL1, a human ubiquitin-like protein associating with human RAD51/RAD52 proteins. Genomics 36, 271-279.

Siliciano, J.D., Canman, C.E., Taya, Y., Sakaguchi, K., Appella, E., and Kastan, M.B. (1997). DNA damage induces phosphorylation of the amino terminus of p53. Genes & development 11, 3471-3481.

Sionov, R.V., Moallem, E., Berger, M., Kazaz, A., Gerlitz, O., Ben-Neriah, Y., Oren, M., and Haupt, Y. (1999). c-Abl neutralizes the inhibitory effect of Mdm2 on p53. The Journal of biological chemistry 274, 8371-8374.

Sorensen, P.H., Lynch, J.C., Qualman, S.J., Tirabosco, R., Lim, J.F., Maurer, H.M., Bridge, J.A., Crist, W.M., Triche, T.J., and Barr, F.G. (2002). PAX3-FKHR and PAX7- FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children's oncology group. J Clin Oncol 20, 2672-2679.

Stahl, M., Dijkers, P.F., Kops, G.J., Lens, S.M., Coffer, P.J., Burgering, B.M., and Medema, R.H. (2002). The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2. J Immunol 168, 5024-5031.

Stein, J.P., Ginsberg, D.A., Grossfeld, G.D., Chatterjee, S.J., Esrig, D., Dickinson, M.G., Groshen, S., Taylor, C.R., Jones, P.A., Skinner, D.G., and Cote, R.J. (1998). Effect of p21WAF1/CIP1 expression on tumor progression in bladder cancer. Journal of the National Cancer Institute 90, 1072-1079.

Storz, P. (2006). Reactive oxygen species-mediated mitochondria-to-nucleus signaling: a key to aging and radical-caused diseases. Sci STKE 2006, re3.

Suliman, A., Lam, A., Datta, R., and Srivastava, R.K. (2001). Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways. Oncogene 20, 2122-2133.

Sun, P., Dong, P., Dai, K., Hannon, G.J., and Beach, D. (1998). p53-independent role of MDM2 in TGF-beta1 resistance. Science 282, 2270-2272.

Sunters, A., Fernandez de Mattos, S., Stahl, M., Brosens, J.J., Zoumpoulidou, G., Saunders, C.A., Coffer, P.J., Medema, R.H., Coombes, R.C., and Lam, E.W. (2003). FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. The Journal of biological chemistry 278, 49795-49805.

- 165 - Suri-Payer, E., and Fritzsching, B. (2006). Regulatory T cells in experimental autoimmune disease. Springer seminars in immunopathology 28, 3-16.

Takata, T., and Ishikawa, F. (2003). Human Sir2-related protein SIRT1 associates with the bHLH repressors HES1 and HEY2 and is involved in HES1- and HEY2-mediated transcriptional repression. Biochemical and biophysical research communications 301, 250-257.

Tan, P., Cady, B., Wanner, M., Worland, P., Cukor, B., Magi-Galluzzi, C., Lavin, P., Draetta, G., Pagano, M., and Loda, M. (1997). The cell cycle inhibitor p27 is an independent prognostic marker in small (T1a,b) invasive breast carcinomas. Cancer research 57, 1259-1263.

Tao, W., and Levine, A.J. (1999). P19(ARF) stabilizes p53 by blocking nucleo- cytoplasmic shuttling of Mdm2. Proceedings of the National Academy of Sciences of the United States of America 96, 6937-6941.

Tatar, M., Kopelman, A., Epstein, D., Tu, M.P., Yin, C.M., and Garofalo, R.S. (2001). A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 292, 107-110.

Thisse, C., Neel, H., Thisse, B., Daujat, S., and Piette, J. (2000). The Mdm2 gene of zebrafish (Danio rerio): preferential expression during development of neural and muscular tissues, and absence of tumor formation after overexpression of its cDNA during early embryogenesis. Differentiation; research in biological diversity 66, 61-70.

Thomas, J.H. (1993). Chemosensory regulation of development in C. elegans. Bioessays 15, 791-797.

Thompson, P.R., Wang, D., Wang, L., Fulco, M., Pediconi, N., Zhang, D., An, W., Ge, Q., Roeder, R.G., Wong, J., Levrero, M., Sartorelli, V., Cotter, R.J., and Cole, P.A. (2004). Regulation of the p300 HAT domain via a novel activation loop. Nature structural & molecular biology 11, 308-315.

Tissenbaum, H.A., and Guarente, L. (2001). Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410, 227-230.

Tomaru, Y., Kondo, S., Suzuki, M., and Hayashizaki, Y. (2003). A comprehensive search for HNF-3alpha-regulated genes in mouse hepatoma cells by 60K cDNA microarray and chromatin immunoprecipitation/PCR analysis. Biochemical and biophysical research communications 310, 667-674.

Tothova, Z., Kollipara, R., Huntly, B.J., Lee, B.H., Castrillon, D.H., Cullen, D.E., McDowell, E.P., Lazo-Kallanian, S., Williams, I.R., Sears, C., Armstrong, S.A., Passegue, E., DePinho, R.A., and Gilliland, D.G. (2007). FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. Cell 128, 325-339. - 166 - Tran, H., Brunet, A., Grenier, J.M., Datta, S.R., Fornace, A.J., Jr., DiStefano, P.S., Chiang, L.W., and Greenberg, M.E. (2002). DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296, 530- 534.

Tsuchida, A., Yamauchi, T., Ito, Y., Hada, Y., Maki, T., Takekawa, S., Kamon, J., Kobayashi, M., Suzuki, R., Hara, K., Kubota, N., Terauchi, Y., Froguel, P., Nakae, J., Kasuga, M., Accili, D., Tobe, K., Ueki, K., Nagai, R., and Kadowaki, T. (2004). Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity. The Journal of biological chemistry 279, 30817-30822.

Uchida, C., Miwa, S., Kitagawa, K., Hattori, T., Isobe, T., Otani, S., Oda, T., Sugimura, H., Kamijo, T., Ookawa, K., Yasuda, H., and Kitagawa, M. (2005). Enhanced Mdm2 activity inhibits pRB function via ubiquitin-dependent degradation. The EMBO journal 24, 160-169.

Urbich, C., Knau, A., Fichtlscherer, S., Walter, D.H., Bruhl, T., Potente, M., Hofmann, W.K., de Vos, S., Zeiher, A.M., and Dimmeler, S. (2005). FOXO-dependent expression of the proapoptotic protein Bim: pivotal role for apoptosis signaling in endothelial progenitor cells. Faseb J 19, 974-976.

Valleix, S., Niel, F., Nedelec, B., Algros, M.P., Schwartz, C., Delbosc, B., Delpech, M., and Kantelip, B. (2006). Homozygous nonsense mutation in the FOXE3 gene as a cause of congenital primary aphakia in humans. American journal of human genetics 79, 358- 364.

Van Der Heide, L.P., Hoekman, M.F., and Smidt, M.P. (2004). The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. The Biochemical journal 380, 297-309. van der Heide, L.P., and Smidt, M.P. (2005). Regulation of FoxO activity by CBP/p300- mediated acetylation. Trends in biochemical sciences 30, 81-86. van der Horst, A., de Vries-Smits, A.M., Brenkman, A.B., van Triest, M.H., van den Broek, N., Colland, F., Maurice, M.M., and Burgering, B.M. (2006). FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP. Nature cell biology 8, 1064-1073. van der Horst, A., Tertoolen, L.G., de Vries-Smits, L.M., Frye, R.A., Medema, R.H., and Burgering, B.M. (2004). FOXO4 is acetylated upon peroxide stress and deacetylated by the longevity protein hSir2(SIRT1). The Journal of biological chemistry 279, 28873- 28879.

Vaziri, H., Dessain, S.K., Ng Eaton, E., Imai, S.I., Frye, R.A., Pandita, T.K., Guarente, L., and Weinberg, R.A. (2001). hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 107, 149-159. - 167 - Voelter-Mahlknecht, S., and Mahlknecht, U. (2006). Cloning, chromosomal characterization and mapping of the NAD-dependent histone deacetylases gene . International journal of molecular medicine 17, 59-67.

Voges, D., Zwickl, P., and Baumeister, W. (1999). The 26S proteasome: a molecular machine designed for controlled . Annual review of biochemistry 68, 1015- 1068.

Vousden, K.H., and Lu, X. (2002). Live or let die: the cell's response to p53. Nature reviews 2, 594-604.

Wang, C., Fu, M., Angeletti, R.H., Siconolfi-Baez, L., Reutens, A.T., Albanese, C., Lisanti, M.P., Katzenellenbogen, B.S., Kato, S., Hopp, T., Fuqua, S.A., Lopez, G.N., Kushner, P.J., and Pestell, R.G. (2001). Direct acetylation of the hinge region by p300 regulates transactivation and hormone sensitivity. The Journal of biological chemistry 276, 18375-18383.

Wang, M.C., Bohmann, D., and Jasper, H. (2005). JNK extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling. Cell 121, 115-125.

Wang, X.W., Zhan, Q., Coursen, J.D., Khan, M.A., Kontny, H.U., Yu, L., Hollander, M.C., O'Connor, P.M., Fornace, A.J., Jr., and Harris, C.C. (1999). GADD45 induction of a G2/M cell cycle checkpoint. Proceedings of the National Academy of Sciences of the United States of America 96, 3706-3711.

Weber, J.D., Taylor, L.J., Roussel, M.F., Sherr, C.J., and Bar-Sagi, D. (1999). Nucleolar Arf sequesters Mdm2 and activates p53. Nature cell biology 1, 20-26.

Wessells, R.J., Fitzgerald, E., Cypser, J.R., Tatar, M., and Bodmer, R. (2004). Insulin regulation of heart function in aging fruit flies. Nature genetics 36, 1275-1281.

Wilkinson, K.D. (1997). Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. Faseb J 11, 1245-1256.

Wilkinson, K.D., Tashayev, V.L., O'Connor, L.B., Larsen, C.N., Kasperek, E., and Pickart, C.M. (1995). Metabolism of the polyubiquitin degradation signal: structure, mechanism, and role of isopeptidase T. Biochemistry 34, 14535-14546.

Wilm, B., James, R.G., Schultheiss, T.M., and Hogan, B.L. (2004). The forkhead genes, Foxc1 and Foxc2, regulate paraxial versus intermediate mesoderm cell fate. Developmental biology 271, 176-189.

- 168 - Wood, J.G., Rogina, B., Lavu, S., Howitz, K., Helfand, S.L., Tatar, M., and Sinclair, D. (2004). Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature 430, 686-689.

Woods, Y.L., and Rena, G. (2002). Effect of multiple phosphorylation events on the transcription factors FKHR, FKHRL1 and AFX. Biochemical Society transactions 30, 391-397.

Wu, J., Shen, Z.Z., Lu, J.S., Jiang, M., Han, Q.X., Fontana, J.A., Barsky, S.H., and Shao, Z.M. (1999). Prognostic role of p27Kip1 and apoptosis in human breast cancer. British journal of cancer 79, 1572-1578.

Xiao, Z.X., Chen, J., Levine, A.J., Modjtahedi, N., Xing, J., Sellers, W.R., and Livingston, D.M. (1995). Interaction between the and the oncoprotein MDM2. Nature 375, 694-698.

Yaciuk, P., and Moran, E. (1991). Analysis with specific polyclonal antiserum indicates that the E1A-associated 300-kDa product is a stable nuclear phosphoprotein that undergoes cell cycle phase-specific modification. Molecular and cellular biology 11, 5389-5397.

Yadav, N., Lee, J., Kim, J., Shen, J., Hu, M.C., Aldaz, C.M., and Bedford, M.T. (2003). Specific protein methylation defects and gene expression perturbations in coactivator- associated arginine methyltransferase 1-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 100, 6464-6468.

Yamamoto, M., Meno, C., Sakai, Y., Shiratori, H., Mochida, K., Ikawa, Y., Saijoh, Y., and Hamada, H. (2001). The transcription factor FoxH1 (FAST) mediates Nodal signaling during anterior-posterior patterning and node formation in the mouse. Genes & development 15, 1242-1256.

Yamamura, Y., Lee, W.L., Inoue, K., Ida, H., and Ito, Y. (2006). RUNX3 cooperates with FoxO3a to induce apoptosis in gastric cancer cells. The Journal of biological chemistry 281, 5267-5276.

Yan, J., Xu, L., Crawford, G., Wang, Z., and Burgess, S.M. (2006). The forkhead transcription factor FoxI1 remains bound to condensed mitotic chromosomes and stably remodels chromatin structure. Molecular and cellular biology 26, 155-168.

Yang, Y., Hou, H., Haller, E.M., Nicosia, S.V., and Bai, W. (2005). Suppression of FOXO1 activity by FHL2 through SIRT1-mediated deacetylation. The EMBO journal 24, 1021-1032.

Yao, K.M., Sha, M., Lu, Z., and Wong, G.G. (1997). Molecular analysis of a novel winged helix protein, WIN. Expression pattern, DNA binding property, and alternative

- 169 - splicing within the DNA binding domain. The Journal of biological chemistry 272, 19827- 19836.

Yao, T.P., Oh, S.P., Fuchs, M., Zhou, N.D., Ch'ng, L.E., Newsome, D., Bronson, R.T., Li, E., Livingston, D.M., and Eckner, R. (1998). Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300. Cell 93, 361-372.

Ye, H., Kelly, T.F., Samadani, U., Lim, L., Rubio, S., Overdier, D.G., Roebuck, K.A., and Costa, R.H. (1997). Hepatocyte nuclear factor 3/fork head homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues. Molecular and cellular biology 17, 1626-1641.

Yogosawa, S., Miyauchi, Y., Honda, R., Tanaka, H., and Yasuda, H. (2003). Mammalian Numb is a target protein of Mdm2, ubiquitin ligase. Biochemical and biophysical research communications 302, 869-872.

You, H., Jang, Y., You-Ten, A.I., Okada, H., Liepa, J., Wakeham, A., Zaugg, K., and Mak, T.W. (2004a). p53-dependent inhibition of FKHRL1 in response to DNA damage through protein kinase SGK1. Proceedings of the National Academy of Sciences of the United States of America 101, 14057-14062.

You, H., Pellegrini, M., Tsuchihara, K., Yamamoto, K., Hacker, G., Erlacher, M., Villunger, A., and Mak, T.W. (2006a). FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal. The Journal of experimental medicine 203, 1657-1663.

You, H., Yamamoto, K., and Mak, T.W. (2006b). Regulation of transactivation- independent proapoptotic activity of p53 by FOXO3a. Proceedings of the National Academy of Sciences of the United States of America 103, 9051-9056.

You, Y., Huang, T., Richer, E.J., Schmidt, J.E., Zabner, J., Borok, Z., and Brody, S.L. (2004b). Role of f-box factor in differentiation of ciliated airway epithelial cells. American journal of physiology 286, L650-657.

Yu, J.K., Holland, N.D., and Holland, L.Z. (2002). An amphioxus winged helix/forkhead gene, AmphiFoxD: insights into vertebrate neural crest evolution. Dev Dyn 225, 289-297.

Yu, Z.K., Geyer, R.K., and Maki, C.G. (2000). MDM2-dependent ubiquitination of nuclear and cytoplasmic P53. Oncogene 19, 5892-5897.

Yuan, Z.M., Huang, Y., Ishiko, T., Kharbanda, S., Weichselbaum, R., and Kufe, D. (1997). Regulation of DNA damage-induced apoptosis by the c-Abl tyrosine kinase. Proceedings of the National Academy of Sciences of the United States of America 94, 1437-1440.

- 170 - Yuan, Z.M., Huang, Y., Whang, Y., Sawyers, C., Weichselbaum, R., Kharbanda, S., and Kufe, D. (1996). Role for c-Abl tyrosine kinase in growth arrest response to DNA damage. Nature 382, 272-274.

Zannini, M., Avantaggiato, V., Biffali, E., Arnone, M.I., Sato, K., Pischetola, M., Taylor, B.A., Phillips, S.J., Simeone, A., and Di Lauro, R. (1997). TTF-2, a new forkhead protein, shows a temporal expression in the developing thyroid which is consistent with a role in controlling the onset of differentiation. The EMBO journal 16, 3185-3197.

Zhang, Y., Wolf, G.W., Bhat, K., Jin, A., Allio, T., Burkhart, W.A., and Xiong, Y. (2003). Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53- dependent ribosomal-stress checkpoint pathway. Molecular and cellular biology 23, 8902-8912.

Zhang, Y., and Xiong, Y. (2001). Control of p53 ubiquitination and nuclear export by MDM2 and ARF. Cell Growth Differ 12, 175-186.

Zhao, Y.Y., Gao, X.P., Zhao, Y.D., Mirza, M.K., Frey, R.S., Kalinichenko, V.V., Wang, I.C., Costa, R.H., and Malik, A.B. (2006). Endothelial cell-restricted disruption of FoxM1 impairs endothelial repair following LPS-induced vascular injury. The Journal of clinical investigation 116, 2333-2343.

Zhou, B.P., Liao, Y., Xia, W., Zou, Y., Spohn, B., and Hung, M.C. (2001). HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nature cell biology 3, 973-982.

Zhou, Y., Kato, H., Asanoma, K., Kondo, H., Arima, T., Kato, K., Matsuda, T., and Wake, N. (2002). Identification of FOXC1 as a TGF-beta1 responsive gene and its involvement in negative regulation of cell growth. Genomics 80, 465-472.

Zhu, W., Bijur, G.N., Styles, N.A., and Li, X. (2004). Regulation of FOXO3a by brain- derived neurotrophic factor in differentiated human SH-SY5Y neuroblastoma cells. Brain research 126, 45-56.

Zhu, Y., Carroll, M., Papa, F.R., Hochstrasser, M., and D'Andrea, A.D. (1996). DUB-1, a deubiquitinating enzyme with growth-suppressing activity. Proceedings of the National Academy of Sciences of the United States of America 93, 3275-3279.

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About the author

Wei Fu received her Bachelor’s degree in Biology from Shaanxi Normal

University in China in 1994 and her Master’s degree in Medical Microbiology and

Immunology from Xi’an Medical University in 1997. During her graduate year she worked in the laboratory of Dr. Yonglie Chu and completed her thesis was on the molecular biology of the human papillomavirus in cervical cancer. Wei worked also as a research assistant in the Molecular Center of the First Clinical Hospital of Xi’an Jiaotong

Univeristy where she carried out both basic and clinical study. Wei started her Graduate

School studies in the fall of 2001 at University of South Florida, Tampa, Florida, and persued her Ph.D thesis work under the tutelage of Dr. Wenlong Bai studying the regulation of FOXO in stress response and tumorigenesis. She successfully defended her doctoral dissertation in November 2007 at the University of South Florida. Wei is a member of the Amercian Cancer Association and the American Heart Association.