Transcript 4th Module,1st Lecture - KSU Faculty Member websites

1st Lecture
Cell Cycle Control, Defects and Apoptosis
Dr Gihan E-H Gawish, MSc, PhD
Molecular Genetics & Clinical Biochemistry
KSU
Cell-Cycle Control in Mammalian Cells
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The Cell Cycle Is an Ordered Series of
Events Leading to Replication of Cells
Regulated Protein Phosphorylation and Degradation Control
Passage through the Cell Cycle
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.
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Regulated Protein
Phosphorylation and
Degradation Control Passage
through the Cell Cycle
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Diverse Experimental Systems Have Been Used
to Identify and Isolate Cell-Cycle Control Proteins
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
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
Isolation of wild-type cell-division cycle (CDC) genes from S.
cerevisiae cells carrying temperature-sensitive mutations in
these genes
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Mammalian Restriction Point is Analogous to
start in Yeast Cells
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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.
Multiple Cdks and Cyclins Regulate Passage of
Mammalian Cells through the Cell Cycle
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Experimental demonstration that cyclin D is required for passage
through the restriction point in the mammalian cell cycle
Multiple Cdks and Cyclins Regulate Passage of
Mammalian Cells through the Cell Cycle
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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.
Passage through the Restriction Point Depends on
Activation of E2F Transcription Factors
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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
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and Cdk2 – cyclin E activity as the cell approaches the G1→S transition.

Mammalian Cyclin-Kinase Inhibitors Contribute to
Cell-Cycle Control
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
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.
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The Rb Protein Acts as a Brake in Mammalian G1 Cells
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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
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back to stimulate the transcription of its own gene, forming another positive feedback loop
Cell-Cycle Progression is Blocked by DNA Damage and p53: DNA Damage
Checkpoints
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When DNA is damaged, protein kinases
that phosphorylate p53 are activated.
Mdm2 normally binds
promotes destruction.
to
p53
and
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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The Major Cell-cycle Regulatory Proteins
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 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 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
SCF
catalyzes 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
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 MCdk activity
Hct1
maintains APC activity after anaphase and throughout G1; inhibited by Cdk activity
Gene regulatory proteins
E2F
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p53
promotes 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
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
Programmed Cell Death (Apoptosis)
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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.
The caspase cascade
involved in apoptosis
(A) Each suicide protease is
made
as
proenzyme
an
inactive
(procaspase),
which is usually activated by
proteolytic cleavage by another
member of the caspase family
(B) Each activated caspase
molecule
can
procaspase
cleave
many
molecules,
thereby activating them, and
these can then activate even
more procaspase molecules.
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Procaspases Are Activated by Binding to Adaptor Proteins
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Bcl-2 Family Proteins Are the Main Intracellular
Regulators of the Cell Death Program
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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.
Dr Gihan Gawish
IAP Proteins Are the Main Intracellular Regulators of
the Cell Death Program
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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
Dr Gihan Gawish
Extracellular Control of Cell Division, Cell
Growth, and Apoptosis
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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.
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.