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Lecture 9

Lecture 9. G1-S. Outline: budding yeast START G1/S in mammalian cells models of cyclin/cdk function. Paper: cyclin knockout/knockin mice.  alternating S- and M-phases no G1 or G2 phases. M. S. early embryonic cell cycle. MPF inactivation leads to DNA replication.

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Lecture 9

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  1. Lecture 9 G1-S Outline: budding yeast START G1/S in mammalian cells models of cyclin/cdk function Paper: cyclin knockout/knockin mice

  2.  alternating S- and M-phases no G1 or G2 phases M S early embryonic cell cycle MPF inactivation leads to DNA replication No other control regulating entry into S phase

  3. Embryonic cell cycle spindle assembly High MPF MPF = Cdk1-Cyclin B Low MPF anaphase cytokinesis DNA replication centrosome duplication

  4. somatic cell cycle varies in length, coupled to growth tightly controlled at G1/S transition point of commitment: yeast  START vertebrates  restriction point

  5. Somatic cell cycle spindle assembly High MPF Low MPF SPF = S phase Promoting factor anaphase cytokinesis Start DNA replication centrosome duplication

  6. regulated by: cell growth nutrient availability signals from other cells G1/S is the major control point: in budding yeast in mammalian tissue culture cells  budding yeast a valuable system

  7. Both YEASTs Cell cycle coordinated with growth at two points: G1/S and G2/M WT fission yeast: cells born large enough to pass start G1/S transition less visible - long G2 WT budding yeast: cells big enough to pass start are big enough to enter mitosis G2/M transition less visible - long G1

  8. committed to divide even if nutrients removed short long

  9. Budding Yeast Cdk1 = CDC28 = cdc2 cdc28 mutations - arrest with no bud analogous to MPF: SPF = S-phase Promoting Factor Cdc28/cyclin heterodimer how to identify cyclin partner? look for high copy suppressors of cdc28ts mutations

  10. CLN1, CLN2 genes identified also CLN3 homology to mitotic cyclins “cyclin box” one CLN gene sufficient triple knockout lethal

  11. Proof that G1 Cyclins control Start Inducible Cln3: To examine cell cycle state: stain with DNA dye pass through fluorescence-activated cell sorter 2 peaks: G1 cells (1N) and G2 cells (2N)

  12. G1 phase short

  13. cln1-/ cln2-/cln3- cells + Cln3 vector Cln necessary and sufficient to induce passage through start

  14. model for progression through G1/S Cdc28-Cln3 induced at proper cell size phosphorylates and activates transcription factors  CLN1 and CLN2, and DNA replication enzymes late G1 CLB5 and CLB6 APC phosphorylated and inactivated  Clb5 and Clb6 (S-phase cyclins) Cdc28-Clb5, Cdc28-Clb6 immediately inactivated by Sic 1

  15. Sic1 CDK inhibitor Induced by Cdc14 phosphatase during exit from mitosis Binds and inhibits S-phase Clb-Cdc28 complexes no effect on G1 Cln-Cdc28 complexes Sic1 degradation causes S-phase onset When phosphorylated byG1 Cln-Cdc28  recognized by ubiquitination machinery SCF = Skp1-cullen-F-box-protein complex  Ubiquitin E3 ligase Analogous to APC

  16. promotes one transition: induces exit from M-phase by inhibiting B-type cyclins inhibits another transition: provides barrier to S-phase that must be overcome Sic1: 2 important roles

  17. Clb5, Clb6 Cln1,2,3 E2 E3

  18. Cln3 Cln1, Cln2 G1 Clb5, Clb6 Clb4, Clb3 S M Clb1, Clb2 Cyclin Expression: Sense cell size Commit to division Activate replication origins Spindle assembly, anaphase

  19. Replication origins fire only once per cell cycle Early G1: pre-replication complexes assemble S-phase: Cdc28-Clb complexes phosphorylate proteins that activate DNA replication  Cdc28-Clb activity prevents assembly of new pre-replication complexes M-phase: Clbs degraded by APC Back to G1

  20. helicase kinase

  21. budding yeast cell cycle

  22. Two modular complexes that direct proteolysis  E3 ubiquitin ligases SCF APC G1/S M exit substrates phosphorylated subunits phosphorylated

  23. What generates specificity? APC Accessory factors: SCF F-box proteins: Cdc20: Clb3, Clb5, Pds1 Cdc4: Sic1 Cdh1: Clb1, Clb2, Clb3 Grr1: Cln1, Cln2

  24. SCF E2 Cullin Ub E3 complex RING Ub S Ub F-box Not just involved in cell cycle transtions  signal transduction proteosome

  25. Cell cycle control in mammalian cells Quiescent cells in G0 phase “mitogens” required to stimulate proliferation = growth factors Multiple Cdks many cloned on the basis of their ability to complement yeast mutations Multiple Cyclins

  26. G1 Cdk4, Cdk6 D Cyclins G1-S Cdk2 Cyclin E S Cdk2 Cyclin A G2/M Cdk1 B Cyclins Phase Cdk Cyclin

  27. induced by mitogens

  28. G1/S D-type cyclin requirement

  29. Cyclin D antibody DNA BrdU Initiation of DNA synthesis blocked by antibody

  30. point of no return = restriction point

  31. What happens downstream of growth factors? early: Fos, Jun delayed: E2F, D Cyclins, Cyclin E, Cdk2, 4, 6

  32. transcription factors activated post-translationally early genes induce transcription of delayed genes

  33. Regulation of E2F transcription factors E2Fs induce: themselves Cdk2 Cyclin E, Cyclin A tumor suppressors E2F Inhibited by retinoblastoma protein (Rb), p107, p130 Rb inhibited by phosphorylation target of Cdk4,6-Cyclin D

  34. positive feedback loop Rb phosphorylation maintained until Cyclin B destruction

  35. Cyclin Kinase Inhibitors (CKI) Two major classes: Cip/Kip: p21, p27, p57 Inhibits Cdk1-, Cdk2-, Cdk4/6-Cyclin complexes Ink4: p16 Inhibits Cdk4/6-D Cyclins

  36. p27Kip1 Inhibits G1/S Phosphorylation by Cdk2-Cyclin E causes degradation by SCF Knockout mouse develops normally, but 30% bigger p57Cip Expressed in differentiating cells Knockout mouse dies, developmental defects: cells fail to become post-mitotic

  37. The CKI swap Cdk4,6-Cyclin D1 activates Cdk2-Cyclin E By phosphorylating Rb By sequestering Cip/Kip If Ink4 induced Displaces Cip/Kip on Cdk4,6-Cyclin D1 Cip/Kip inhibits Cdk2-Cyclin E

  38. i n k c i p E D1 i n k Cdk2 Cdk4,6 a mechanism to halt G1/S progression D1 c i p Cdk4,6 X S-phase

  39. E2F Cyclin A Cdh1 Cyclin B Cdk2-cyclin A Required to progress through S phase Required for cyclin B accumulation phosphorylates and inactivates Cdh1  blocks cyclin B degradation by proteosome

  40. What generates Cdk/cyclin substrate specificity? How are diverse events induced? Cdk subunit? Only slight differences in substrate specificity Both Cdk1 and Cdk2 can rescue cdc28 mutations Cyclin subunit? Some mediate interaction with substrates Cyclin D-Rb Different affinity for inhibitors But: A single cyclin can allow fission yeast to grow normally

  41. Cdk2 + Cyclin A

  42. Qualitative model for Cdk/Cyclin function

  43. Quantitative model for Cdk/Cyclin function

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