2. Dr Gihan E-H Gawish, MSc, PhD Molecular Genetics & Clinical Biochemistry KSU Cell Cycle Control, Defects and Apoptosis 1 st Lecture

3. In vertebrates and diploid yeasts, cells in G 1 have a diploid number of chromosomes (2n), one inherited from each parent. In haploid yeasts, cells in G 1 have one of each chromosome (1n). Rapidly replicating human cells progress through the full cell cycle in about 24 hours: mitosis takes ≈30 minutes; G 1, 9 hours; the S phase, 10 hours; and G 2, 4.5 hours. In contrast, the full cycle takes only ≈90 minutes in rapidly growing yeast cells. 2 Cell-Cycle Control in Mammalian Cells The Cell Cycle Is an Ordered Series of Events Leading to Replication of Cells

4. Dr Gihan Gawish 3  the complex macromolecular events of the eukaryotic cell cycle are regulated by a small number of heterodimeric protein kinases.  Passage through three critical cell-cycle transitions, is irreversible because these transitions are triggered by the regulated degradation of proteins, an irreversible process. As a consequence, cells are forced to traverse the cell cycle in one direction only. Regulated Protein Phosphorylation and Degradation Control Passage through the Cell Cycle

5. Dr Gihan Gawish 4 Regulated Protein Phosphorylation and Degradation Control Passage through the Cell Cycle

6.  Amphibian and invertebrate eggs and early embryos from synchronously fertilized eggs provide sources of extracts for biochemical studies of cell-cycle events.  The isolation of yeast cell-division cycle (cdc) mutants led to the identification of genes that regulate the cell cycle Diverse Experimental Systems Have Been Used to Identify and Isolate Cell-Cycle Control Proteins 5

7. Dr Gihan Gawish 6 Isolation of wild-type cell-division cycle (CDC) genes from S. cerevisiae cells carrying temperature-sensitive mutations in these genes

8. 7  In multicellular organisms, cell replication is controlled by a complex network of signaling pathways that integrate signals from the extracellular environment with intracellular cues about cell size and developmental program.  Polypeptide growth factors called mitogens stimulate cultured mammalian cells to cycle. Once cycling cells pass the restriction point, they can enter the S phase and complete S, G 2, and mitosis in the absence of growth factors. Mammalian Restriction Point is Analogous to start in Yeast Cells

9. 8 Multiple Cdks and Cyclins Regulate Passage of Mammalian Cells through the Cell Cycle Experimental demonstration that cyclin D is required for passage through the restriction point in the mammalian cell cycle

10. 9 Multiple Cdks and Cyclins Regulate Passage of Mammalian Cells through the Cell Cycle Activity of mammalian Cdkcyclin complexes through the course of the cell cycle in G 0 cells induced to divide by treatment with growth factors The width of the colored bands is approximately proportional to the protein kinase activity of the indicated complexes. Cyclin D refers to all three D-type cyclins.

11. Dr Gihan Gawish 10 Regulation of Rb and E2F activities in late G 1  Stimulation of G 0 cells with mitogens induces expression of Cdk4, Cdk6, D-type cyclins and E2F transcription factors (E2Fs), all encoded by delayed-response genes.  Interaction of E2Fs with hypophosphorylated Rb protein initially inhibits E2F activity.  When signaling from mitogens is sustained, the resulting Cdk4 – cyclin D and Cdk6 – cyclin D complexes (Cdk4/6 – cyclin D) initiate the phosphorylation of Rb, converting some E2F to the active form.  Active E2F then stimulates its own synthesis and the synthesis of Cdk2 and cyclin E.  Cdk2 – cyclin E further stimulates Rb phosphorylation releasing more E2F activity. These processes result in positive feedback loops (blue arrows) leading to a rapid rise in both E2F and Cdk2 – cyclin E activity as the cell approaches the G 1 → S transition. Passage through the Restriction Point Depends on Activation of E2F Transcription Factors

12. Dr Gihan Gawish 11  Mammalian cells are thought to express a cyclin-kinase inhibitor (CKI) that functions like S. cerevisiae Sic1.  Mammalian cells are known to express several CKIs that contribute to cell-cycle control.  These are grouped into two classes:  CIP (Cdk inhibitory protein) family bind and inhibit all Cdk1-, Cdk2-, Cdk4-, and Cdk6-cyclin complexes  INK4 (inhibitors of kinase 4) family bind and inhibit only Cdk4 – cyclin D and Cdk6 – cyclin D complexes. Mammalian Cyclin-Kinase Inhibitors Contribute to Cell-Cycle Control

13. Dr Gihan Gawish 12 Mechanisms controlling S-phase initiation in animal cells G 1 -Cdk activity (cyclin D-Cdk4) initiates Rb phosphorylation. This inactivates Rb, freeing E2F to activate the transcription of S-phase genes, including the genes for a G 1 /S-cyclin (cyclin E) and S-cyclin (cyclin A). The resulting appearance of G 1 /S-Cdk and S-Cdk activities further enhances Rb phosphorylation, forming a positive feedback loop. E2F acts back to stimulate the transcription of its own gene, forming another positive feedback loop Mechanisms controlling S-phase initiation in animal cells G 1 -Cdk activity (cyclin D-Cdk4) initiates Rb phosphorylation. This inactivates Rb, freeing E2F to activate the transcription of S-phase genes, including the genes for a G 1 /S-cyclin (cyclin E) and S-cyclin (cyclin A). The resulting appearance of G 1 /S-Cdk and S-Cdk activities further enhances Rb phosphorylation, forming a positive feedback loop. E2F acts back to stimulate the transcription of its own gene, forming another positive feedback loop

14. Dr Gihan Gawish 13 When DNA is damaged, protein kinases that phosphorylate p53 are activated. Mdm2 normally binds to p53 and promotes destruction. Phosphorylation of p53 blocks its binding to Mdm2; as a result, p53 accumulates to high levels and stimulates transcription of the gene that encodes the CKI protein p21. The p21 binds and inactivates G 1 /S-Cdk and S-Cdk complexes, arresting the cell in G 1. In some cases, DNA damage also induces either the phosphorylation of Mdm2 or a decrease in Mdm2 production, which causes an increase in p53 When DNA is damaged, protein kinases that phosphorylate p53 are activated. Mdm2 normally binds to p53 and promotes destruction. Phosphorylation of p53 blocks its binding to Mdm2; as a result, p53 accumulates to high levels and stimulates transcription of the gene that encodes the CKI protein p21. The p21 binds and inactivates G 1 /S-Cdk and S-Cdk complexes, arresting the cell in G 1. In some cases, DNA damage also induces either the phosphorylation of Mdm2 or a decrease in Mdm2 production, which causes an increase in p53

15. 14 The Cell-Cycle Control System

16. 15 GENERAL NAMEFUNCTIONS AND COMMENTS Protein kinases and protein phosphatases that modify Cdks Cdk-activating kinase (CAK) phosphorylates an activating site in Cdks Wee1 kinasephosphorylates inhibitory sites in Cdks; primarily involved in controlling entry into mitosis Cdc25 phosphatase removes inhibitory phosphates from Cdks; three family members (Cdc25A, B, C) in mammals; Cdc25C is the activator of Cdk1 at the onset of mitosis Cdk inhibitory proteins (CKIs) Sic1 (budding yeast)suppresses Cdk activity in G 1 ; phosphorylation by Cdk1 triggers its destruction p27 (mammals) suppresses G 1 /S-Cdk and S-Cdk activities in G 1 ; helps cells to withdraw from cell cycle when they terminally differentiate; phosphorylation by Cdk2 triggers its ubiquitylation by SCF p21 (mammals)suppresses G 1 /S-Cdk and S-Cdk activities following DNA damage in G 1 ; transcriptionally activated by p53 p16 (mammals)suppresses G 1 -Cdk activity in G 1 ; frequently inactivated in cancer Ubiquitin ligases and their activators SCF catalyzes ubiquitylation of regulatory proteins involved in G 1 control, including CKIs (Sic1 in budding yeast, p27 in mammals); phosphorylation of target protein usually required for this activity APC catalyzes ubiquitylation of regulatory proteins involved primarily in exit from mitosis, including Securin and M-cyclins; regulated by association with activating subunits Cdc20 APC-activating subunit in all cells; triggers initial activation of APC at metaphase-to- anaphase transition; stimulated by M- Cdk activity Hct1maintains APC activity after anaphase and throughout G 1 ; inhibited by Cdk activity Gene regulatory proteins E2F promotes transcription of genes required for G 1 /S progression, including genes encoding G 1 /S cyclins, S-cyclins, and proteins required for DNA synthesis; stimulated when G 1 -Cdk phosphorylates Rb in response to extracellular mitogens p53 promotes transcription of genes that induce cell cycle arrest (especially p21) or apoptosis in response to DNA damage or other cell stress; regulated by association with Mdm2, which promotes p53 degradation The Major Cell-cycle Regulatory Proteins

17. 16  The number of cells in this community is tightly regulated—not simply by controlling the rate of cell division, but also by controlling the rate of cell death process known as programmed cell death, or apoptosis.  Apoptosis is mediated by proteolytic enzymes called caspases, which trigger cell death by cleaving specific proteins in the cytoplasm and nucleus.  Caspases exist in all cells as inactive precursors, or procaspases, which are usually activated by cleavage by other caspases, producing a proteolytic caspase cascade.  The activation process is initiated by either extracellular or intracellular death signals, which cause intracellular adaptor molecules to aggregate and activate procaspases. Caspase activation is regulated by members of the Bcl-2 and IAP protein families.  The number of cells in this community is tightly regulated—not simply by controlling the rate of cell division, but also by controlling the rate of cell death process known as programmed cell death, or apoptosis.  Apoptosis is mediated by proteolytic enzymes called caspases, which trigger cell death by cleaving specific proteins in the cytoplasm and nucleus.  Caspases exist in all cells as inactive precursors, or procaspases, which are usually activated by cleavage by other caspases, producing a proteolytic caspase cascade.  The activation process is initiated by either extracellular or intracellular death signals, which cause intracellular adaptor molecules to aggregate and activate procaspases. Caspase activation is regulated by members of the Bcl-2 and IAP protein families. Programmed Cell Death (Apoptosis)

18. Dr Gihan Gawish 17 (A) Each suicide protease is made as an inactive proenzyme (procaspase), which is usually activated by proteolytic cleavage by another member of the caspase family (B) Each activated caspase molecule can cleave many procaspase molecules, thereby activating them, and these can then activate even more procaspase molecules. (A) Each suicide protease is made as an inactive proenzyme (procaspase), which is usually activated by proteolytic cleavage by another member of the caspase family (B) Each activated caspase molecule can cleave many procaspase molecules, thereby activating them, and these can then activate even more procaspase molecules. The caspase cascade involved in apoptosis

19. Dr Gihan Gawish 18 Procaspases Are Activated by Binding to Adaptor Proteins

20. Dr Gihan Gawish 19 Bcl-2 Family Proteins Are the Main Intracellular Regulators of the Cell Death Program The Bcl-2 family of intracellular proteins helps regulate the activation of procaspases. Some members of this family, like Bcl-2 itself or Bcl-XL, inhibit apoptosis, at least partly by blocking the release of cytochrome c from mitochondria. Other members of the Bcl-2 family are not death inhibitors, but instead promote procaspase activation and cell death. Some of these apoptosis promoters, such as Bad, function by binding to and inactivating the death-inhibiting members of the family, whereas others, like Bax and Bak, stimulate the release of cytochrome c from mitochondria.

21. Dr Gihan Gawish 20 IAP Proteins Are the Main Intracellular Regulators of the Cell Death Program they bind to some procaspases to prevent their activation, and they bind to caspases to inhibit their activity IAP proteins were originally discovered as proteins produced by certain insect viruses, which use them to prevent the infected cell from killing itself before the virus has had time to replicate. When mitochondria release cytochrome c to activate Apaf-1, they also release a protein that blocks IAPs, thereby greatly increasing the efficiency of the death activation process

22. 21 The factors that promote organ or organism growth can be operationally divided into three major classes:  Mitogens, which stimulate cell division, primarily by relieving intracellular negative controls that otherwise block progress through the cell cycle.  Growth factors, which stimulate cell growth (an increase in cell mass) by promoting the synthesis of proteins and other macromolecules and by inhibiting their degradation.  Survival factors, which promote cell survival by suppressing apoptosis. Extracellular Control of Cell Division, Cell Growth, and Apoptosis The extracellular signal molecules that regulate cell size and cell number are generally either soluble secreted proteins, proteins bound to the surface of cells, or components of the extracellular matrix.