Dr Gihan E-H Gawish, MSc, PhDMolecular Genetics & Clinical Biochemistry

KSU

Cell Cycle Control, Defects and Apoptosis

1st Lecture

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In vertebrates and diploid yeasts,

cells in G1 have a diploid number of

chromosomes (2n), one inherited

from each parent.

In haploid yeasts, cells in G1 have

one of each chromosome (1n).

Rapidly replicating human cells

progress through the full cell cycle

in about 24 hours: mitosis takes ≈30

minutes; G1, 9 hours; the S phase, 10

hours; and G2, 4.5 hours. In contrast,

the full cycle takes only ≈90 minutes

in rapidly growing yeast cells.

Cell-Cycle Control in Mammalian Cells

The Cell Cycle Is an Ordered Series of Events Leading to Replication of Cells

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Dr Gihan Gawish

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

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Dr Gihan Gawish

Regulated Protein Phosphorylation and

Degradation Control Passage through the Cell Cycle

5 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

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Dr Gihan Gawish

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

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 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, G2, and mitosis in the

absence of growth factors.

Mammalian Restriction Point is Analogous to start in Yeast Cells

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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

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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 G0 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.

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Dr Gihan Gawish

Regulation of Rb and E2F activities in late G1

Stimulation of G0 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 G1→S transition.

Passage through the Restriction Point Depends on Activation of E2F Transcription

Factors

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Dr Gihan Gawish

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

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Dr Gihan Gawish

The Rb Protein Acts as a Brake in

Mammalian G1 Cells

Mechanisms controlling S-phase initiation in animal cells

G1-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 G1/S-cyclin (cyclin E) and S-cyclin (cyclin A). The resulting

appearance of G1/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

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Dr Gihan Gawish

Cell-Cycle Progression is Blocked by DNA Damage and p53:

DNA Damage Checkpoints

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 G1/S-Cdk and S-Cdk complexes, arresting the cell in G1. In some cases, DNA damage also induces either the phosphorylation of Mdm2 or a decrease in Mdm2 production, which causes an increase in p53

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The Cell-Cycle Control System

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GENERAL NAME FUNCTIONS AND COMMENTS

Protein kinases and protein

phosphatases that modify Cdks

Cdk-activating kinase (CAK)

phosphorylates an activating site in Cdks

Wee1 kinase phosphorylates inhibitory sites in Cdks; primarily involved in controlling entry into mitosis

Cdc25 phosphataseremoves 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 G1; phosphorylation by Cdk1 triggers its destruction

p27 (mammals)suppresses G1/S-Cdk and S-Cdk activities in G1; helps cells to withdraw from cell cycle when they terminally differentiate; phosphorylation by Cdk2 triggers its ubiquitylation by SCF

p21 (mammals) suppresses G1/S-Cdk and S-Cdk activities following DNA damage in G1; transcriptionally activated by p53

p16 (mammals) suppresses G1-Cdk activity in G1; frequently inactivated in cancer

Ubiquitin ligases and their activators

SCFcatalyzes ubiquitylation of regulatory proteins involved in G1 control, including CKIs (Sic1 in budding yeast, p27 in mammals); phosphorylation of target protein usually required for this activity

APCcatalyzes ubiquitylation of regulatory proteins involved primarily in exit from mitosis, including Securin and M-cyclins; regulated by association with activating subunits

Cdc20APC-activating subunit in all cells; triggers initial activation of APC at metaphase-to- anaphase transition; stimulated by M-Cdk activity

Hct1 maintains APC activity after anaphase and throughout G1; inhibited by Cdk activity

Gene regulatory proteins

E2Fpromotes transcription of genes required for G1/S progression, including genes encoding G1/S cyclins, S-cyclins, and proteins required for DNA synthesis; stimulated when G1-Cdk phosphorylates Rb in response to extracellular mitogens

p53promotes 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

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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)

17 Dr Gihan Gawish

(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

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Procaspases Are Activated by Binding to Adaptor Proteins

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Dr Gihan Gawish

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.

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Dr Gihan Gawish

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

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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.