Discovery of MPF (M-Phase Promoting Factor)

Transcription

1 Discovery of MPF (M-Phase Promoting Factor) transfert de cytoplasme après GVBD Dettlaff, 1964 Bufo sp. GVBD GVBD Masui & Markert, 1971 ; Smith & Ecker, 1971 Rana pipiens GVBD GVBD Kishimoto, 1976 Etoile de mer GVBD GVBD

2 Universality of the MPF Factor Kishimoto, 1978 Starfish GVBD GVBD Bufo sp.

3 Cytoplasmic factor and Meiotic Maturation

4 Molecular Characterization of MPF Regulatory subunit Cyc B Cyc B cdk1 Wee1/Myt1 Cyc B cdk1 MPF inactive cdk1 P Y15 P Thr14 Catalytic subunit CAK Cdk1=Cdc2 P Thr161 Cyc B cdk1 Thr14 P P Y15 MPF inactive Cdc25 P Thr161 Cyc B cdk1 MPF active

5 The auto-amplification loop? G2 Cdc25 Inactive Mitosis Plx Thr14 P P P P Thr161 Cdc25 active P P P P Cdk1 Cdk1 Tyr15 Cyclin B Inactive MPF Wee 1 Myt 1 active Cyclin B active MPF Thr161 Mitotic Entry Wee 1 Myt 1 P P Inactive

6 The Cdk-Cyclin complexes during the Cell Cycle

7 Cyclin B Degradation P P Cdc25 Cdc25 Cyc B cdk1 Protein kinase active P Thr161 Myt1 P P Wee 1 Wee 1 Cyc B cdk1 Thr14 P P P Tyr 15 Thr161 Protein kinase inactive cdk1 inactive cdk1 inactive Cyclin B Translation

8 Cyclin B Degradation require an active D-box sequence NH 2 COOH R-T-V-L-G-V-I-G-D R-T-A-L-G-N-I-G-N R-A-A-L-G-E-I-G-N Cyclin A Cyclin B1 Cyclin B2 NH 2 COOH X*-T-A-L-G-N-I-G-N Cycline B1 undegradable Mitotic Arrest High CdK-Cyclin B Activity Cyclin B Degradation Pathway ON

9 Proteins degradation by the 26S proteasome CONJUGAISON DE L'UBIQUITINE DEGRADATION DES PROTEINES AMP ATP iquitine () E1- E1 AMINO- ACIDES E2- E2(s) ATP PEPTIDES PROTEINE ADP E3 ATP PROTEASOME 26S Présentation des antigènes Enzymes de dé-ubiquitination + Complexe 19S (PA700) Protéasome 20S 2002, Dr. Thierry LORCA, CRBM-CNRS, Montpellier

10 THE DISCOVERY OF APC (Anaphase Promoting Complex) NH2 D Box E2 APC/E3 U K K C COOH P U E2 E1 Cdk1-cyclin B Proteasome 26S NH2 D Box U U U U K K C COOH U U U U

11 Cyc B cdk1 P Thr161 Kinase active min A B C ( 35 S)-Cyc. B Cyc B Thr161 P cdk1 Multiple ubiquitin conjugates ( 35 S)-Cyc. B 1. Degradation in presence of the protéasome 26S 2. Inactivation of the cdk1-cyclin B kinase 3. Mitotic Exit

12 Activation of APC during the Cell cycle Cdc20/Fizzy Cdh1/Fizzy-R Cdc20/Fizzy APC Inactive APC active Cdc20/Fizzy APC active Cdh1/Fizzy-R Métaphase Anaphase Mitotic exit Securin Degradation Cyclin B degradation

13 activé APC Cdh1/Fizzy-R Cdh1/Fizzy-R iquitination and Degradation of Protein during mitotic exit APC active Cdh1/Fizzy-R APC activé MOTIF Protein Protein U b Motifs: - D-Box : RxxL - KEN Box : KEN APC Inactive Cdh1/Fizzy-R Protéasome 26 S Degradation

14 APC Regulation Cdc20/Fizzy Cyclin B cdk1 APC Inactif P M APC active Cdc20/Fizzy Cdh1/Fizzy-R Cdc20/Fizzy G1 APC active Fizzy Cdh1/Fizzy-R R. Polo PKA Cyclin B cdk1 P Métaphase Anaphase Mitotic Exit

15 E3 Ligases et Cell Cycle! 1 substrat! F-box Skp1 cullin Roc1 Rbx1 SCF Complex Ring finger domain 2 substrat! Cdc20/Cdh1 Cdc23/16 APC11 Cdc27 Ring finger domain APC Complex substrat! Chfr/! Siah! Ring Finger E3 3 Ring finger domain substrat! Cdc23/16 APC11 Cdc27 Ring finger domain

16 2. Relationship between SCF et APC! G2 Mitosis G1 Cyclin B! P! Cdk1! P! P! Inactive Cdc25 wee1 Cyclin B! Cdk1! P! Active ΤΟΜΕ-1 SCF TOME-1 wee1 Wee1 Proteolysis cdh1 TOME-1 Proteolysis APC active P!

17 Two Physiological pathways regulate the Metaphase-Anaphase Transition

18 The spindle checkpoint detects Mitotic Spindle Defects SOMATIC CELL UNATTACHED KINETOCHORE Metaphase Mitosis exit Mitosis arrest Interphase The spindle checkpoint arrests Cell division before separation of Sister Chromatids

19 Spindle mitotic checkpoint in yeast Nocodazole or benomyl Mitotic checkpoint activation Metaphase arrest Genes: MAD-1, MAD-2, MAD-3 (Mitotic Arrest Deficient) BUB-1, BUB-2, BUB-3 (Budding Uninhibited by Benomyl) MPS1 (Mono-Polar-Spindle)

20 BUB-1 and MPS1 are Protein Kinases Bub-1 or Mps1 overexpression induces a metaphase arrest Mps1 and Bub-1 phosphorylate Mad1 and Bub-3 in vitro, respectively Formation of complexes MAD-1/MAD-2 BUB-1/BUB-3 BUB-1/BUB-3/MAD-1

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23 Metaphase-Anaphase transition requires Securin proteolysis Separase APC Cdc20 Separase Proteasome + Separase Microtubule Separase Cohesin Cohesin Co sin Kinetochore Cohesin Complex Metaphase Anaphase

24 X-Mps1 controls the association of CENP-E and the Mad1/Mad2 complex with kinetochores + Sperm nuclei +!Nocodazole Mad2 CENP-E Merge w/ Mad1 CENP-E Merge w/ (Texas red) (FITC) DAPI (Texas red) (FITC) DAPI IgG Depletion X-Mps1 Depletion 2002, Dr. Thierry LORCA, CRBM-CNRS, Montpellier

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26 Meiotic maturation in xenopus oocytes Metaphase II arrest Interphase Maturation Fertilization

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37 Meiotic maturation in xenopus oocytes Metaphase II arrest Interphase Maturation Fertilization 2002, Dr. Thierry LORCA, CRBM-CNRS, Montpellier

38 Protéolyse de EMI 2 Fécondation cdc20 APC Emi2 cdc20 Emi2 Cycline B! Cdk1! P! cdc20 Emi2 P Active APC cdc20 dégradation de la Cycline B Sortie de Meiose/mitose Plx DSGxxS motif P! P! Emi2 P P! P! Emi2 βtrcp SCF P EMI 2 est dégradée après la fécondation

39 The END