Regulation and functions of protein kinase B in
DNA damage signaling
Erlangung der Würde eines Doktors der Philosophie
der Universität Basel
Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel. Im Auftrag von Dr. Brian A. Hemmings, Prof. Dr. Markus Affolter, Prof. Dr. Matthias Wymann and Dr. Ruth Chiquet-Ehrismann.
Prof. Dr. Markus Affolter Prof. Dr. Matthias Wymann
Basel, den 9.12. 2008.
Prof. Dr. Eberhard Parlow
List of Abbreviations
Ataxia Telangiectasia Mutated ATM Ataxia Telangiectasia and Rad3 Related ATR Damage Response DDR DNA
DNA Double Strand Breaks DNA DSBs
DNA Dependent Protein Kinase Catalytic Subunit DNA-PK cs
Forkhead box, Class O FOXO Glycogen Synthase Kinase 3 GSK3 Insulin-like Growth Factor 1 IGF1 Insulin Receptor Substrate IRS Mammalian Target of Rapamycin mTOR
3-phosphoinositide-dependent Protein Kinase 1 PDK1 Pleckstrin Homology PH Phosphoinositide-3-kinase PI3K Phosphatidylinositol 3,4,5-triphosphate (PIP) PI(3,4,5)P3 3
Protein Kinase B PKB Phosphatase and Tensin homolog PTEN
Regulatory-associated Protein of mTOR (mTORC1 complex) Raptor Rapamycin-insensitive Companion of mTOR (mTORC2 complex) Rictor Receptor Tyrosine Kinase RTK PI3K-related Kinase PIKK
Less frequently used abbreviations are defined upon their first use in the text.
Table of Content
1. SUMMARY 1
2. INTRODUCTION 5
2.1. Cellular stress signaling and the PI3-kinase-like kinases (PIKKs) 7
2.1.1. PI3-kinase-like family of protein kinases (PIKKs) 7
2.1.2. Nutrient signaling 11
2.1.3. Genome surveillance signaling 14
184.108.40.206. DNA-dependent protein kinase (DNA-PK) structure,
activity and functions 17
2.2. Protein kinase B (PKB) 23
2.2.1. PKB isoforms and structure 23
2.2.2. Regulation of PKB activity by phosphorylation 25
2.2.3. Physiological functions of PKB 28
220.127.116.11. PKB substrates and functions 28
18.104.22.168. Genetic systems to elucidate PKB signaling 31
2.3. SCOPE OF THE THESIS 35
3. RESULTS AND DISCUSSION 37
3.1. DNA-PK AND mTOR DYNAMICALLY REGULATE PKB PHOSPHORYLATION AND
ACTIVATION UNDER DIFFERENT CELLULAR CONDITIONS 39
3.1.1. Summary 40
3.1.2. Results 41
3.1.3. Discussion 45
3.2. PKB； ACTS DOWNSTREAM OF DNA-PK IN THE DNA DOUBLE-STRAND BREAK RESPONSE AND PROMOTES SURVIVAL 47
3.2.1. Summary 48
3.2.2. Published manuscript 49
PKBalpha/Akt1 Acts Downstream of DNA-PK in the DNA Double-Strand Break Response and Promotes Survival
Lana Bozulic, Banu Surucu, Debby Hynx and Brian A. Hemmings Mol Cell. 30(2):203-13
3.2.3. Supplementary figures 62
3.3. EXPLORING THE POSSIBILITES FOR THERAPEUTIC INTERVENTION: SPECIFIC INHIBITION OF PI3K； IN THE DNA-DAMAGE RESPONSE 80
3.3.1. Introduction 81
3.3.2. Summary and preliminary results 83
4. GENERAL DISCUSSION AND CONCLUSIONS 88
5. REVIEWS 102
5.1. Targeting the Kinome: 20 Years of Tyrosine Kinase Inhibitors in Basel.
6. BIBLIOGRAPHY 114
7. APPENDIX 132
7.1. In Vivo Analysis of Protein Kinase B (PKB)/Akt Regulation in DNA-PK-cs
null Mice Reveals a Role for PKB/Akt in DNA Damage Response and Tumorigenesis
Surucu B, Bozulic L, Hynx D, Parcellier A, Hemmings BA (2008) J Biol Chem. 283(44):30025-33
8. CURRICULUM VITAE 145
Conservedfrom primitive metazoans to humans, protein kinase B (PKB or Akt) is a serine/threonine kinase with well-characterized functions in a number of essential cellular processes such as transcription, proliferation and survival. Its activity is regulated via phosphorylation of the conserved residues, Thr308 in the activation loop, and Ser473 in the hydrophobic motif. This results in a 10x and 1000x increase in PKB kinase activity, respectively. Clearly, deregulation of its activity can contribute to development of pathogenic states such as diabetes or cancer. Hence much recent PKB research has focused on the identification and characterization of the PKB hydrophobic motif Ser473 kinase.
We investigated the specific contributions of three PI3-kinase-like family members (PIKKs) in PKB Ser473 phosphorylation. These included DNA-dependent protein kinase (DNA-PK), mammalian target of rapamycin complex 2 (mTORC2) and ataxia-telangiectasia-mutated (ATM), as work from our and other laboratories proposed these kinases as PKB regulators. Stable inducible RNAi system was employed to specifically deplete TRex293 cells of respective kinases. We found that in growing cells (cycling cells cultured in fully supplemented medium), the integrity of mTORC2 is necessary for PKB Ser473 phosphorylation. When cells were starved (cultured in serum-free medium), and subsequently mitogen stimulated, both mTOR and DNA-PK contributed to the activation of PKB, which was reflected by PKB downstream signaling. Finally, DNA-PK was required for ，-IR-induced PKB phosphorylation/activation. We found that ablation of ATM by RNAi has no effect on PKB activity in any of the conditions tested. We concluded that mTOR and DNA-PK have overlapping and distinct physiological
roles in regulating PKB activity, these being determined by the incitement or challenge to the cells.
Further, we investigated in more detail PKB activation following DNA damage. This required 3-phosphoinositide-dependent kinase 1 (PDK1) and DNA-dependent protein kinase (DNA-PK). Active PKB localized in the nucleus of ，-
irradiated cells adjacent to DNA double-strand breaks, where it co-localized and interacted with DNA-PK. Levels of active PKB inversely correlated with DNA damage-induced apoptosis. Accordingly, a significant portion of p53- and DNA damage-regulated genes were misregulated in cells lacking PKB；. Lastly, PKB；
knock-out mice showed impaired DNA damage-dependent induction of p21 and increased tissue apoptosis after single dose total body irradiation. Our findings place PKB downstream of DNA-PK in the DNA damage response signaling cascade, where it provides a pro-survival signal, in particular by affecting transcriptional p21 regulation. Furthermore, this function is apparently restricted to the PKB； isoform.
Together, our results reveal a complex pattern of PKB regulation in response to various stimuli. This is achieved by PKB Ser473 phosphorylation being carried out by (at least) two different enzymes. Furthermore, this also possibly enables a context-dependent result of PKB activation, as we describe its specific roles in DNA damage signaling where active PKB promotes survival.