Cell Cycle Regulation II Study Guide PDF

Biochemical foundations of pharmaceutical biotechnology Cell Cycle Regulation II and Drugs that Target the Cell Cycle Audrey Minden, Ph.D. Department of Chemical Biology Ernest Mario School of Pharmacy Susan Lehman Cullman Laboratory for Cancer Research, Room 205 (848) 445-5766 [email protected]...

Biochemical foundations of pharmaceutical biotechnology Cell Cycle Regulation II and Drugs that Target the Cell Cycle Audrey Minden, Ph.D. Department of Chemical Biology Ernest Mario School of Pharmacy Susan Lehman Cullman Laboratory for Cancer Research, Room 205 (848) 445-5766 [email protected] Lectures 1-10: (Exam 1) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. The importance of Biochemistry and Molecular Biotechnology in drug development (Molloy) Basic cell biology and cell architecture (Minden) An overview of the science of large biomolecules (Minden) Control of cell behavior A: Cell membrane and transport, drugs that impact cell transport (Minden) Control of cell behavior B: Signal transduction-1 (Minden) Signal transduction-2 and drugs that interfere with signal transduction pathways (Minden) Cell cycle regulation (Minden) Cell cycle regulation part B and drugs that target the cell cycle (Minden) Cancer: Normal cells vs cancer cells (Minden) Oncogenes and their inhibition in managing cancer (Minden) 2 Cell Cycle II. Outline of topics covered: I. History of the discovery of MPF A. Studies in xenopus oocytes B. Studies in yeast C. Studies in sea urchin D. Studies in mammalian cells II. Purification of MPF III. The functions of MPF at M phase A. Cdc2 and Cyclin B are the main components B. Substrates of Cdc2 C. Regulation of MPF IV. Cyclins and CDKs (and Cdcs) in the cell cycle A. Regulation of Cyclins and CDKs B. The role for CKIs V. Regulation of Cyclin D / CDK4//6 A. Rb B. p53 C. TGF-beta D. Drugs that target CDK4/6 3 What is the molecular basis of cell cycle regulation? Some history on the identification of MPF, a critical protein complex in cell cycle regulation 4 Identification of MPF To understand the molecular mechanism by which the cell cycle is regulated, studies were done in the following cell types: Frog (Xenopus) oocytes, Yeast, Sea urchin eggs, and Mammalian cells. 5 Studies with Xenopus oocytes: 6 Xenopus oocytes Oocytes go through meiosis arrest (figure modified from libretexts.org) 7 Cytoplasmic injection studies with Xenopus oocytes M phase arrested cell G2 phase arrested cell M phase cell 8 Conclusions from Xenopus studies: Some factor in the cytoplasm of M phase cells can cause the G2 arrested cells to enter M phase. This factor was referred to as MPF (Maturation Promoting Factor) MPF (figure modified from libretexts.org) 9 Yeast studies Different types of yeast: Saccharomyces cerevisiae (budding yeast) and Saccharomyces pombe (fission yeast). 10 Identification of cdc (cell division cycle) mutants and the corresponding genes in yeast, by using yeast genetics studies Cdc28 (gene in saccharomyces cerevisiae) Cdc2 (gene in saccharomyces pombe) These two genes were found in the two yeast strains, to be important for progression from G1 to S 11 Conclusions from yeast studies: Certain genes encode proteins that are required for the yeast to pass through various phases of the cell cycle. These proteins are called cdcs. (later in mammalian cells they are named cdks) They are later found to be important components of MPF. 12 Studies with sea urchin eggs: 13 Identification of Cyclins in sea urchin eggs (Cells were grown in the presence of radiolabeled amino acids) (Figure from Hunt, T. 2011) 14 Conclusions from Sea Urchin Studies - Some proteins in the sea urchin egg show a cyclic pattern throughout the cell cycle. - These are called Cyclins. - The were later found to be components of MPF 15 Studies with mammalian cells: Cytoplasm injection experiments using mammalian cells Mammalian cell arrested in M phase Frog oocyte arrested at G2 Now the oocyte goes through M phase (Inject cytoplasm) 16 Conclusions from studies in mammalian cells: - Some factor in the cytoplasms of M phase arrested cells can cause the G2 arrested cells to enter M phase. - This factor is highly conserved, as it can cross species. - It is referred to as MPF (M-phase Promoting Factor) MPF 17 Purification and characterization of MPF from Xenopus 18 MPF M phase arrested cell G phase arrested cell M phase cell Scientists purified the cytoplasmic factor (named MPF) that caused the G phase cells to enter M phase 19 Purification and characterization of MPF from Xenopus MPF was found to contain two components a) A 34 kd protein b) A 45 kd protein 20 Purification and characterization of MPF from Xenopus The 45 kd protein turned out to be a homologue of Cyclin B The 34 kd protein turned out to be a homologue of cdc2 Cyclin B MPF cdc2 (Cdc2 stands for Cell Division Cycle protein 2. Later it was renamed CDK1, for Cyclin Dependent Kinase 1) MPF activity cycles throughout the cell cycle This type of complex is highly conserved: found in yeast, xenopus, sea urchin, mammals, … 21 MPF: Cyclin plus CDC/CDK How does the MPF complex regulate the cell cycle? 22 MPF consists of cyclin B and Cdc2 cyclin B Cdc2 Cdc2 (CDK1) is a protein kinase It depends on a cyclin (cyclin B) for its activity 23 What does MPF do? MPF cyclin B 2 Cdc2 1. Cdc2 (CDK1) phosphorylates substrates that are important for M phase 2. Consequently, the cell goes into M phase cyclin B 1 Active cyclin B Cdc2 3 Cdc2 M G2 Cdc2 G1 3. Cdc2 phosphorylates Cyclin B, which then gets degraded Inactive protein kinase S cyclin B 4. The cycle can start over again when more Cyclin B is made Active protein kinase Cdc2 4 cyclin B 24 Cdc2 (CDK1) substrates: - Nuclear lamins. These are a type of nuclear protein, comparable to the cytoskeleton. Phosphorylation of nuclear lamins causes the nuclear lamina to break down during mitosis. This is followed by nuclear membrane breakdown. - Histone H1 (HH1). HH1 plays a role in packaging DNA into chromosomes. Phosphorylation may help induce chromosome condensation. - MAPs. Microtubule Associated Proteins: Play a role in re-organization of microtubules. They bind to tubulin and have a role in regulating microtubule stability 25 ( P = phosphate groups ) How is MPF regulated? d. A phosphatase Cyclin B dephosphorylates two of Cdc2 Thr-161 the phospho-sites, by P Thr-14 Tyr-15 a phosphatase. Active Now MPF is active. CDC25 Activated MPF can family M of phosphatases now phosphorylate substrates and G2 Cyclin B promote M phase Inactive Cdc2 Thr-161 Important: this is P Thr-14 Tyr-15 P the active form S P of MPF Cyclin B e. After M phase, Cdc2 phosphorylates Cyclin B and targets it for destruction Cyclin B + Cdc2 c. Cdc2 is phosphorylated on three regulatory protein positions, by two kinaseskinases Thr-14 Cdc2 Thr-161 P Thr-14 Tyr-15 Inactive Cdc2 G1 Thr-14 Thr-161 Tyr-15 Inactive A b. Cyclin B/Cdc2 complex forms Thr-161 Tyr-15 Inactive Cyclin B a. Cyclin B synthesis begins in S phase Protein kinase How is MPF regulated? f. In some cases a CKI (cyclin Dependent ( P = phosphate groups ) kinase inhibitor) can inhibit the complex d. A phosphatase Cyclin B dephosphorylates two of CKI Cdc2 Thr-161 the phospho-sites, by P Thr-14 Tyr-15 a phosphatase. Active Now MPF is active. CDC25 Activated MPF can family M of phosphatases now phosphorylate substrates and G2 Cyclin B promote M phase Inactive Cdc2 Thr-161 Important: this is P Thr-14 Tyr-15 P the active form S P of MPF Cyclin B e. After M phase Cdc2 phosphorylates Cyclin B and targets it for destruction Cyclin B + Cdc2 c. Cdc2 is phosphorylated on three regulatory protein positions, by two kinaseskinases Thr-14 Cdc2 Thr-161 P Thr-14 Tyr-15 Inactive Cdc2 G1 Thr-14 Thr-161 Tyr-15 Inactive Examples of CKIs: p21, p16,… A b. Cyclin B/Cdc2 complex forms Thr-161 Tyr-15 Inactive Cyclin B a. Cyclin B synthesis begins in S phase Protein kinase Different cyclins and CDKs operate at different stages of the cell cycle in eukaryotic cells CDKs and cyclins are parts of families of proteins that operate at different parts of the cell cycle Example in mammalian cells: Cyclin B Cdk1 Cyclin A Cdk1 Cyclin D (D1, D2, D3) Cdk2, 4, 6 Cyclin E Cdk2 28 Cyclin D Rb P53 TGF-beta 29 Cyclin D / CDK 2,4,6 Another important cyclin is Cyclin D. It operates at the G1/S transition growth factors Cyclin B Cdk1 Cyclin D (D1, D2, D3) Cyclin A Cdk1 CDK 2, 4, 6 Cyclin E Cdk2 30 Rb 31 Rb is a key substrate of Cyclin D1-Cdk4/6 Rb DP1 P M Rb E2F DP1 Rb G2 growth factors E2F G1 Go CyclinD Rb P CyclinD Cdk4/6 S Cdk4/6 Rb restriction point DP1 E2F P Rb P Rb interphase + E2F transcription repressed DP1 DP1 E2F transcription 32 Rb regulates cell cycle progression by regulating transcription factor complex E2F/DP1 DP1 Rb E2F DP1 E2F X Transcription (of genes important for DNA replication, cell cycle progression, ) DP1 E2F Rb P Repression of transcription When Rb is phosphorylated it can not bind E2F Transcription (of genes important for cell cycle progression) 33 p53 34 p53 is a transcription factor. It stimulates p53 levels expression of the gene increase encoding p21, a CKI p53 DNA damage p53 p21 gene p21 mRNA p21 (CKI) Cyclin p21 protein p21 (CKI) Cdk Thr- P Thr+ Tyr- P P Inactive: No progression from G1 to S 35 P53 can inhibit cell cycle progression by inhibiting the cyclinD/CDK complex (via the CKI p21) R b P M DNA damage p53 DP 1 Rb E2 F p21 DP 1 Rb G2 Growth Factors E2 F G1 Go Rb P CyclinD CyclinD S Cdk4/6 Rb P restriction point DP1 Rb E2F P Rb Cdk4/6 interphase + transcription repressed E2 F DP 1 DP1 E2F transcription 36 Anti cancer drugs that inhibit CDKs CDK4/6 inhibitors Administration of CDK4/6 inhibitors is usually in combination with hormone therapies to treat adults with hormone receptor (HR) positive, HER2 negative advanced or metastatic breast cancer that has metastasized. CDK 4/6 inhibitors have been shown to improve progression-free survival in metastatic breast cancer. (HR can refer to ER or PR : estrogen or progesterone) Ibrance (palbociclib) is a CDK4/6 inhibitor, Marketed by Pfizer Ibrance is one of Pfizers top selling drugs. It is used for HR+, Her2- metastatic breast cancer, but is currently being tested on other types of cancer. Several other CDK4/6 inhibitors have also been developed and are being used to treat HR+, Her2- breast cancer. 37 Resources: Alberts Molecular Biology of the Cell. Chapter 17 Hunt T. (2011). Getting in and out of mitosis. Rambam Maimonides Medical Journal. 2:E0051 38