TABLE OF CONTENTS I. INTRODUCTION

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TABLE OF CONTENTS

I. INTRODUCTION...1

I.1 The cell cycle...1

I.1.1 History of cell cycle...1

I.1.2 Cell cycle and its phases...2

I.1.3 Regulation of cell cycle progression...3

I.1.4 Key proteins of the cycle...3

I.1.4.1 CDKs...3

I.1.4.2 Cyclins...4

I.1.4.3 CKIs (CDK inhibitors)... 5

I.1.5 Regulation of cyclin-CDK complexes...6

I.2 Regulation of G1 phase...9

I.2.1 The restriction point (R)... 9

I.2.2 The key proteins for G1 phase regulation and for the passage through the restriction point...10

I.2.2.1 The retinoblastoma family...10

I.2.2.2 The E2F family...12

I.2.2.3 Cyclins D...13

I.2.2.4 CDK4 and CDK6...16

I.2.2.5 p21 and p27 CKIs...19

I.3 Cell cycle and cancer...25

I.4 The CDK activating kinases (CAKs)... 28

I.4.1 Is CDK7 the only animal CDK activating kinase?... 30

I.5 c-Jun N-terminal protein kinases (JNKs)... 33

I.5.1 JNKs in cancer...39

II. AIM OF THE WORK AND STRATEGIES...41

III. MATERIALS AND METHODS...43

III.1 Cell cultures...43

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III.2 Transfections...45

III.3 JNK inhibition...45

III.4 Measurement of DNA synthesis...46

III.5 Immunoprecipitations...46

III.6 Measurement of pRb-kinase activity of CDK4 complexes...47

III.7 In vitro phosphorylation of CDK4 complexes and p21 by Cyclin H-CDK7-Mat1, JNK1/2/3, ERK1/2, p38α...49

III.8 Protein separation by monodimensional electrophoresis...49

III.9 Protein separation by bidimensional electrophoresis...50

III.10 Protein immunodetection...52

IV. RESULTS...53

- “CDK4 T172 phosphorylation is central in a CDK7-dependent bi-directional CDK4/2 interplay mediated by p21 phosphorylation at the restriction point” ...54

- “JNKs function as CDK4-activating kinases by phosphorylating CDK4 and p21”... 58

V. DISCUSSION AND CONCLUSIONS...63

- Role of CDK4 in cell cycle...63

- CDK7 implication in CDK4 phosphorylation and activation...64

- Role of p21 and its phosphorylations in CDK4 regulation...65

- A new model for CDK4 activation and passage through the R point...66

- New CDK4 activating kinases...67

- UPDATE: a new model for CDK4 activation and passage through the R point...69

- Perspectives and Conclusions...70

VI. APPENDIX...72

VI.1 CDK4 T172-phosphorylation is central in a CDK7-dependent bidirectional CDK4/2 interplay mediated by p21 phosphorylation at the restriction point...72

VI.2 The CDK4/6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes...73

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VII. BIBLIOGRAPHY ...76

- Abstract (french and english)