Cell Cycle

1. Yeast Cell Cycle • Cell cycle mechanisms are universal, irrespective of the species from which the cell is derived and the biological context of its division. Cells in culture Frog oocytes

2. Cell Cycle Control The principle: • Cell cycle progression must occur in a particular sequence. This sequence must be preserved even if steps take longer. The cellcycle control system achieves this by molecular brakes that can stop the cycle at various check points. • These check points are also points where the control system can be regulated by extracellular factors.

3. M Proceed through mitosis G1 G2 interphase Trigger DNA replication machinery S Cell Cycle Control Prophase Metaphase Anaphase Telophase Trigger mitosis machinery Metaphase check point: chromosome alignment mitosis G2 checkpoint: cell size environment DNA replication G1 checkpoint: cell size environment How is the cell cycle regulated?

4. Protein phosphorylation: The most common post-translational modification that regulates biological processes tyrosine, threonine and serine are subjected to phosphorylation on their free hydroxyl groups. 85% 15% < 0.01% Concept: change in structure leads to a change in function

5. Isolation of the Key Proteins:Frog oocytesYeast

6. Cell Cycle Control: Isolation of Mitosis Promoting Factor (MPF) Driven to M phase (spindle detected) Inject cytoplasm from M phase cell Does not enter M phase Inject cytoplasm from interphase cell Injected extracts: from developing frog eggs, rapid cell cycles injected cells: oocytes- cells arrested before first meiotic division

7. Temperature sensitive (TS) Yeast grown at 25oC Screen for genes complementing defect Grow Yeast at 35 oC GENE Z ISOLATE GENE Z ISOLATION OF CELL DIVISION CYCLE GENES FROM YEAST – the principle Temperature sensitive (TS) Yeast can divide at 25oC but not at 35oC

8. Cell Cycle Control: Two families of proteins • cyclin dependent protein kinases • (CDK; Ser/Thr kinases) • termed cdc for cell division cycle mutants characterized in Yeast • cyclins • have no enzymatic activity of their own, but activate the kinases. Their levels change in a cyclic fashion active form is a dimer How is the activity of the complex regulated?

9. The Proteasome • Large complex of proteolytic enzymes present in the cytosol and in the nucleus - degrade tagged proteins. • What is the tag? • What is the recognition mechanism? Figure 3-18, MCB 4th ed.

10. The “Tag” - Ubiquitin • Originally isolated from the thymus (Goldstein 1975), was thought to be a hormone. Later - found in all tissues. • Found to be part of a branched protein (Goldknopf and Busch 1977). • Intracellular protein degradation was found to require energy (ATP). Using a cell free system, ubiquitin, a protein of 76 amino acids was identified (Ciechanover 1978). • Ligation of ubiquitin to a protein marks it for degradation by a protease that specifically recognizes ubiquitinylated proteins (Hershko Ciechanover and Rose 1979)

11. 25 YEARS LATER- Nobel Prize in Chemistry A. Ciechanover, A. Hershko I. Rose

12. The Ubiquitin-Proteasome System ubiquitin ATP ubiquitin AMP E1 E1 polyubiquitination E2 E3 proteasome ATP ADP peptides • Most intracellular proteins are degraded in large protein complexes - the proteasomes. • Proteasomes degrade proteins that have been marked for degradation by ubiquitin binding.

13. Substrate Specificity of the Ubiquitinating Enzymes Specificity of ubiquitination is determined by interaction of the E3 enzyme with its substrate. Complexity!!!!!

14. Mitosis promoting factor (MPF = G2/M, mitotic CDK/cyclin) MPF=G2/M CDK/cyclin mitosis interphase How is degradation initiated/regulated

15. Control of entry into anaphase and exit from mitosis Anaphase promoting complex (APC), a ubiquitin E3 ligase, is activated by MPF. APC polyubiquitinates the anaphase inhibitor, which is then degraded. The APC polyubiquitinates cyclin B – MPF degradation and exit from mitosis

16. Substrates of MPF (CDK/cyclin) • Protein kinase activity causes radical changes that will lead the cell to mitosis: Phosphorylated substrates: • Lamin on Ser residues; leads to disassembly of the nuclear envelope(advantage?) • Histone H1, (chromosome condensation?) • Microtubule associated proteins and others! • Progression of cell cycle causes timed degradation of the cyclins and inactivation of the CDKs.

17. Several CDK/cyclin complexes regulate passage through the cell cycle CDK/cyclin (MPF) Mitosis CDK/cyclin CDK/cyclin

18. Cdc – Cell division cycle single proteins isolated from Yeast cyclins CDK phosphatases kinases MPF- mitosis promoting factor (G2/M) CDK and cyclin in complex

19. p21 p27 Regulation of CDKs

20. The Biological Context • Growth factor binds to cell surface receptor initiating a cascade of events. • Among activated genes are essential components of the cell cycle control system including CDKs and cyclins, probably involved in driving cells past G1 into S. • These stimulatory signals have to overcome inhibitory devices which ensure proliferation arrest in the absence of signal.

21. More Nobel prize awards….. The 2001 Nobel Prize in Physiology or Medicine 8 October 2001 jointly to Leland H. Hartwell (checkpoints),R. Timothy (Tim) Hunt(CDK)and Paul M. Nurse (cyclins) for their discoveries of "key regulators of the cell cycle" Hunt Hartwell Nurse

22. Perry MC: The Chemotherapy Source Book; Wiliams & Wilkins, 1992; p.500

23. Novel strategies to inhibit cell cycle progression cyclin CDK p21 p27 • Inhibit CDKs: flavopiridol, roscovitine, BMS-387032. • Inhibit proteasome: velcade (bortezomib, PS314) • Stabilize growth inhibitory molecules: eg. P53. (P53 binds to DNA and leads to transcription of p21)

24. Cyclins and CDKS in development and cancer: lessons from genetically modified mice Santamaria andOrtega Front Biosci. 2006. From yeast to humans, cell cycle progression and cell division are driven by the sequential activation of a group of serine-threonine kinases called cyclin-dependent kinases. Multiple Cdks control the cell cycle in mammals and have been long considered essential for normal proliferation, development and homeostasis. The importance of the Cdk-cyclin complexes in cell proliferation is underscored by the finding that deregulation of the Cdk activity is found in virtually the whole spectrum of human tumors. Recent information from gene-targeted mouse models for the various cyclins and Cdks have made some of the generally accepted concepts of cell cycle regulation to be revised and new and exciting questions to be investigated. Unexpectedly, most of the Cdk-cyclin complexes have turned out to be dispensable for cell proliferation due to a high level of functional redundancy, promiscuity and compensatory mechanisms..…This review will discuss these new findings and their implications for cancer therapy

25. The Proteasome — An Emerging Therapeutic Target in Cancer New Eng. J. Med 348:2597-2598 June 2003

27. Bortezomib (Velcade, PS-341) p21 p27 • Augments apoptotic response to chemotherapeutic agents. • Enhances stabilization of p21 and p27 (inhibitors of cyclin/CDK), as well as p53. • Inhibits NF-kappaB, a transcription factor that increases the production of growth factors, cell-adhesion molecules, and anti-apoptotic factors.

28. velcade Inhibition of NF-kappa B