M. Tyers December 6 2006 tyers@mshri.on,ca

1. BCH 2021 Cell Cycle Lecture M. Tyers December 6 2006 tyers@mshri.on,ca

2. 2001 Nobel Prizes for Cell Cycle Lee Hartwell Tim Hunt Paul Nurse what were the key ideas and experiments? check out http://nobelprize.org for bios

3. Cyclin dependent kinases are hetromultimers, composed of catalytic, activating and inhibitory subunits (yeast protein names indicated)

4. Maturation Promoting Factor (MPF)

5. Properties of MPF

6. G1 M S G2 Genetics identified the Cdc2/Cdc28 protein kinase S. pombe = fission yeast (Nurse) S. cerevisiae = budding yeast (Hartwell) cdc2ts at 37oC cdc28ts at 37oC

7. Cyclin - discovered in clams and sea urchins via the synchronous post-fertilization cell cycles to follow 35S-labeled proteins (Hunt et al., 1983) - cyclin oscillates with a peak in M phase; this is due to continuous synthesis and then rapid and complete destruction at the end of mitosis.

8. CDK activity oscillates through the cell cycle: low in G1 phase and high in S/G2/M

9. Structural basis of CDK regulation CDK cyclin

10. Regulatory tyrosine phosphorylation by the Wee1 kinase and Cdc25 phosphatase … and activation by CDK activating kinase (CAK), which is not regulated

11. CDK Inhibitors (CKIs) p27KIP1

12. The ubiquitin system regulates the cell cycle (Hershko, Ciechanover, Rose - 2004 Nobel Prize) G1-S switch M-G1 switch protein degradation cyclins, CKIs are primary substrates

13. Multiple levels of regulation control CDK activity ON:OFF = 107 fold!

14. APC/C SCF Anaphase Promoting Complex/ Cyclosome Skp1 Cdc53/Cullin F-box protein Complex G1 cyclins (Cln-Cdc28) M cyclins (Clb-Cdc28) Cdk inhibitor (Sic1) anaphase inhibitor (Pds1) S cyclins (Clb-Cdc28) G1 CDK OFF CDK ON M S G2 M/G1 switch G1/S switch 2 E3 enzymes establish alternation of CDK activity, which controls origins of DNA replication origins load origins fire origin re-loading blocked

15. APCCdh1 CDK antagonists P P P P MCMs MCMs P P DNA POL P P CDK activity fires replication and then prevents re-replication Start (Cln-Cdc28) CDK antagonists Sic1 Sic1 APCCdh1 CDK (Clb-Cdc28) CDK P P P P P P Cdc6 Cdc6 Cdc6 MCMs Cdc6 ORC ORC ORC origins fire in S phase origin loading blocked until end of mitosis origin loading in G1 phase Mitotic Exit Network (Cdc14 release)

16. Failure to inactivate CDKs causes genome instability Normal CDK inactivation: wild type ≈ 50 kb origin barrier Incomplete CDK inactivation: sic1 deletion chromosome loss improper replication/elevated DNA damage (see Lengronne and Schwob, 2002)

17. unattached kinetochore Mad1/2/3 The M/G1 Switch: The Anaphase Promoting Complex/Cyclosome, a Cullin-RING E3 enzyme

18. Stepwise action of APC/C in mitosis checkpoints MEN APC/C arrest b/c Pds1 not degraded (cdc16, cdc20, cdc23...) APC/CCdc20 APC/CCdh1 Pds1 Clb Esp1 anaphase elongated spindle separated sisters Cdk high Sic1 unstable telophase spindle disassembled cytokinesis occurs re-entry in G1 Cdk low Sic1 stable G2/M short spindle unseparated sisters cohesion MEN arrest b/c Cdc14 not released (cdc5, cdc15, tem1...)

19. 1 2 The Mitotic Exit Network (MEN) cortex reviewed in: Bardin and Amon, 2002 terminal phenotype SPB + bud neck nucleolus

20. Release of the Cdc14 phosphatase from the nucleolus triggers mitotic exit

21. How is mitotic exit network activated? Location...  Tem1-Lte1 interaction senses completion of anaphase Tem1 - GTPase Lte1 - exchange factor

22. Cdc14 is an anti-CDK phosphatase that reactivates the CDK antagonists specifically dephosphorylated in vitro Clb-Cdc28 Cdc14 txn factor Swi5 SIC1 (M/G1 genes) Cdh1 APC/C activator Sic1 CKI Clb-Cdc28 Visintin et al. 1998

23. CDK inactivation is controlled by the MEN

24. The G1/S Switch pheromone nutrients ploidy START = commitment point (restriction point)  critical cell size G1 M S G2 • bud emergence • SPB duplication • DNA replication • cyclin dependent • kinases ON L. Hartwell

25. Transcriptional control of Start Whi3 Cln3- Cdc28 transcription factors G1 cyclin dependent kinases Whi5 Bck2 Start Pheromone G R O W T H ? Bud emergence DNA replication SPB duplication CDK on, APC off SBF/MBF Cln1/2- Cdc28 G1/S transcription ~ 120 genes

26. ~800 genes are cell cycle regulated in budding yeast

27. Transcriptional circuits enable CDK oscillation

28. The CDK OFF state: the inhibitor Sic1 is present only in G1 phase cells Sic1-GFP

29. A threshold of G1 cyclin (Cln)-Cdc28 activity is needed to degrade Sic1 and pass Start SCFCdc4 DNA replication Cln-Cdc28 activity B. Futcher, F. Cross

30. The SCFCdc4 cullin-RING ligase targets Sic1 growth scaffold Cln3-Cdc28 adapter E3 RING-H2 Cln1/2-Cdc28 recruitment (F-box) DNA replication Clb5/6-Cdc28 wild type cdc4/34/53 cdc4/34/53 sic1 1 N 2 N

31. E3 substrate How is Sic1 proteolysis "switched on" at START? phosphorylation-dependent substrate recognition, but unlike SH2 or other domains P see Nash et al, Nature 414, 514 (2001)

32. Cdc4 bound pSic1, 40% 6p.1 5p Sic1 6p.2 3p 2p 3p 7p 2p 5p 9p 0p 6p.1 6p.2 (most potent sites added back first) GAL1-SIC1 ON GAL1-SIC1 OFF 7p 9p At least 6 of 9 CDK phosphorylation sites are required for efficient Sic1 recognition and degradation S76 T2/5 T33 T45 S69 S80 T173 S191 Sic1 1 2 5 3 4 8 7 6 rank genetic effects 9

33. No obvious consensus in Cdc4 substrates (except CDK sites = S/T-P-X-K/R) Sic1 Cdc6 T2 /5 M TP S TP P R S R T7 SAIPI TP TK R I T33 MQGQK TP Q K PS T23 DDAPA TP P R PL T45 NLVPV TP STT K T39 QFTDV TP ESSP S69 NMGMT SP FNGL S43 VTPES SP E K LQ S76 FNGLT SP Q R SP T135 PLSLS TP R S K D S80 TSPQR SP FP K S S354 KRFLL SP T R GS T173 KDVPG TP SD K V T368 AQVPL TP TTSP S191 NWNNN SP K NDA S372 LTPTT SP V KK S also Far1 (14 sites), Gcn4 (5 sites), Ash1 (28 sites) phosphopeptides based on natural sites do not bind Cdc4!

34. How are multiply phosphorylated substrates recognized by Cdc4? Possible Models: 1. Numerous low affinity binding sites simultaneously engage multiple phosphorylated residues 2. Phosphorylation driven conformational change leads to exposure of a cryptic binding epitope 3. Single binding site interacts with multiple phosphorylated residues (low affinity) in equilibrium

35. P P T380 T380 cyclin E cyclin E Cdk2 Sic1pT45 peptide Skp1 alone No peptide 40% Input + ++ +++ CycE-pT380 +++ +++ CycE-T380 pSic1 pCycE Bound to Cdc4-Skp1 Resin A cyclinE-pT380 phosphopeptide out-competes substrate binding but Sic1phosphopeptides do not hCdc4/Ago/ Fbw7/SEL-10 (Reed, Roberts, Hariharan, Haber, Elledge, Harper)

36. A SPOTS peptide array based on CycET380 defines the Cdc4 Phospho-Degron (CPD) Cdc4 Phospho-Degron (CPD): I/L-I/L/P-pT-P-(RK)-(RK)-(RK)-(RK) (Basic Residues Disfavored) VS CDK Consensus: pS/T-P-X-K/R (Basic Residues Preferred)  all Sic1 sites are low affinity probed with anti-Skp1/Cdc4

37. P P P P P P P P P P Optimal CPD versus Sub-Optimal CPD Cdc28 kinase (xxPVTPxK/Rx)n xxLLTPxxx ??? Sic1 Cdc4 Cdc4 Degradation Degradation  but why bother with multiple sites?

38. P A single optimal CPD site (LLTPP) is sufficient for Cdc4 binding and degradation in vivo xxLLTPxxx Sic1CPD T45T S76S - - + - - + - - + Cdc4 - + + - + + - + + kinase LLT76PP T45::CycE Sic1 WT GAL1-SIC1 ON (all in lethal 9 site mutant) Sic1WT Sic1CPD Sic1T45,S76

39. However, Sic1CPD levels are heterogeneous in G1 phase cells (GFP fusions)

40. Sic1 is eliminated immediately in mothers and with a delay in daughters (i.e., longer growth requirement in daughters)

41. Precocious elimination of Sic1CPD (accelerated timing in daughter vs mother)

42. G2 G2 G1 Which results infailure to restrain DNA replication... SIC1 SIC1CPD G1

43. WT G1 sic1 G1 … and genome instability SIC1 SIC1CPD sic1D chromosome loss rate < 1 in 105 100 X increase (rampant)

44. The Skp1-Cdc4-CPD structure cyclin E peptide GLLpTPPQSG

45. rigid interface vs. slim connecting stalk Skp1-Cdc4-CPD modeled on the holo-SCF complex SCFCdc4 SCFhSkp2 ? E2 LRR 59Å 50Å

46. Conserved residues in the CPD binding site cancer mutations (Hariharan, Haber, Reed) basic H polar

47. A basic patch might account for +2 to +5 K/R anti-selection: WT Cdc4 K402A/R443D Can the threshold be altered? I/L-I/L/P-pT-P-(RK)-(RK)-(RK)-(RK) (Basic Residues Disfavored)

48. T2/5 MTPSTPPRSR T33 MQGQKTPQKPS T45 NLVPVTPSTTK S69 NMGMTSPFNGL S76 FNLGTSPQRSP S80 TSPQRSPFPKS T173 KDVPGTPSDKV S191 NWNNNSPKNDA Re-engineering Cdc4 selectivity at the P-1 and P+2 to P+5 positions + – IEF # sites phosphorylated Sic1 sites: 0 1 2 3 4 5 6 7 8 9 input wild type P+2 to P+5 K402A R443D V384N W717N P-1 (capture onto Cdc4 resin) consensus: I/L-I/L/P-pT-P-(RK)-(RK)-(RK)-(RK) Orlicky et al 2003

49. SCFCdc4 Cln-Cdc28 activity Summary The Cdc4 Phospho-Degron (CPD) I/L-I/L/P-pT-P-(rky)-(rky)-(rky)-(rky) is at odds with the CDK consensus Multiple sub-optimal CPDs in Sic1 establish a minimum CDK-free window needed for assembly of replication origins and genome stability But why multi-site phosphorylation?  to set a high threshold  to precisely build a threshold  ultrasensitivity

50. Recall Xenopus maturation: How do oocytes decide when to mature? How is this decision made irreversible? Maturation depends on a MAPK cascade: PG Cdk mos Mek1/2 p42/44Erk1/2 MAPKKK MAPKK MAPK ~ only 30X  4 X1011 1011 3 X109 # of molecules: