Dr Mohammad S Alanazi, MSc, PhDMolecular Biology

KSU

Cell Cycle Control, Defects and Apoptosis

1st Lecture

2

The Cell Cycle Is an Ordered

Series of Events

Leading to Replication of

Cells

Cell-Cycle Control in Mammalian Cells

3

Dr Gihan Gawish

Regulated Protein Phosphorylation and

Degradation Control Passage through the Cell Cycle

4 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

Dr Gihan Gawish

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

6

 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

7

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

8

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.

9

Dr Gihan Gawish

Cell-Cycle Progression is Blocked by

DNA Damage and p53: DNA Damage

Checkpoints

10

The Cell-Cycle Control System

11

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

12

In multicellular organisms, cells that are no longer needed or are a threat to the

organism are destroyed by a tightly regulated cell suicide 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)

13 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

14 Dr Gihan Gawish

Procaspases Are Activated by Binding to Adaptor Proteins

15

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.