The document discusses regulation of the cell cycle through cyclins, CDKs, and ubiquitin ligases. It covers the four phases of the cell cycle (G1, S, G2, M), regulation of DNA replication and mitosis, checkpoints that ensure quality control, and mechanisms that propel quiescent cells into the cell cycle in response to mitogens. The precise ordering and timing of cell cycle events is controlled by the periodic synthesis and degradation of cyclins and other regulatory proteins.

1. Chap. 19 The Eukaryotic Cell Cycle
Topics
• Overview of the Cell Cycle and Its Control
• Molecular Mechanisms for Regulating M & S Phase Events
• Mitogen-stimulated Entry of Cells into the Cell Cycle
• Surveillance Mechanisms in Cell-cycle Regulation
Goals
• Learn the roles of 1) cyclins and cyclin-
dependent protein kinases (CDKs), & 2)
ubiquitin-protein ligases in regulation of
the cell cycle.
• Learn the molecular mechanisms for
regulation of mitosis and S-phase events.
• Learn how mitogens propel quiescent cells
into the cell cycle.
• Learn how checkpoint mechanisms ensure
quality control in cell cycle events.
Cell division during C. elegans
early embryogenesis

2. Two fundamental processes occur with each cell cycle--
chromosomes replicate, and then they segregate equally to two
daughter cells. The mechanisms by which these processes occur
are similar in all eukaryotic cells. Processes occurring during the
cell cycle are highly regulated and coordinated. The cell cycle is
regulated primarily at the DNA replication and mitosis steps.
The master controllers of the cell cycle are 1) heterodimeric
protein kinases composed of a regulatory subunit (a cyclin) and
a catalytic subunit (a cyclin-dependent kinase, CDK), 2) two
ubiquitin-protein ligases, and 3) regulatory phosphatases.
Cyclin-CDKs phosphorylate and thereby regulate the activities
of numerous cell proteins that participate in replication and
division. The bound cyclins regulate the activities of the CDKs.
Ubiquitin-protein ligases participate in the timed destruction of
cyclins and other key proteins and thereby ensure passage
through the cell cycle is irreversible. In the absence of
regulation, cells replicate and divide uncontrollably, leading to
diseases such as cancer.
Overview of the Cell Cycle and Its Control

3. Regulating Protein Function by Degradation
The proteolytic degradation (turnover) of proteins is important for
regulatory processes, cell renewal, and disposal of denatured and
damaged proteins. Lysosomes carry out degradation of endocytosed
proteins and retired organelles.
Cytoplasmic protein degradation
is performed largely by the
molecular machine called the
proteasome. Proteasomes
recognize and degrade
ubiquinated proteins (Fig.
3.29). Ubiquitin is a 76-amino-
acid protein that after
conjugation to the protein,
targets it to the proteasome.
In ATP-dependent steps, the
C-terminus of ubiquitin is
covalently attached to a lysine
residue in the protein.
Polyubiquitination then takes
place. The proteasome
degrades the protein to
peptides, and released ubiquitin
molecules are recycling.

4. Major Events in the Cell Cycle
The cell cycle proceeds via four
phases in cycling (replicating)
somatic cells. These phases are
designated the G1, S, G2, and M
phases (Fig. 19.1). In G1 phase,
cells synthesize many of the
proteins that will be used for
DNA synthesis and chromosome
replication during S phase. G2
follows S and is a transitional
period preceding M phase. M
phase is a multistage period
wherein chromosomes separate
and the cell divides. In a dividing
mammalian cell, the four phases
of the cell cycle typically require
9 h, 10 h, 4.5 h, and 30 min
respectively. Many cells in adult
multicellular organisms do not
proliferate and never, or at least
rarely, divide. These cells exit
the cell cycle in G1 phase and
enter a quiescent phase called G0.

5. Review of M Phase Processes (I)
From an ultrastructural standpoint, M phase processes are the
most complex. In comparison, few changes are visibly apparent in
most cells during interphase, which consists of the combined G1,
S, and G2 phases. M phase is subdivided into 4 main periods--
prophase, metaphase, anaphase, and telophase (Fig. 18.36). In
prophase, replicated chromosomes condense and become visible.
In prometaphase, the nuclear membrane retracts and the mitotic
apparatus known as the spindle forms. Kinetochores assemble at
centromeres and attach the chromosomes to the mitotic spindle
fibers. In metaphase, chromosomes line up on the metaphase
plate in the center of the spindle.

6. Review of M Phase Processes (II)
In anaphase, sister chromatids of each duplicated chromosome
separate and are drawn toward the two spindle poles. Then in
telophase, the mitotic spindle disassembles, chromosomes
decondense, the nuclear envelope reforms surrounding the
chromosomes, and the cell undergoes cytokinesis--the physical
division of the cytoplasm.

7. Mechanism of Cell Cycle Regulation (I)
The molecular mechanisms by which the cell cycle is controlled
in a typical eukaryotic cell is presented in Fig. 19.30 below.
The initiation of the cell cycle occurs with the receipt of a
signal (e.g., a growth factor ligand) by a cell in G0 or G1. The
signal induces synthesis of G1 and G1/S phase cyclin-CDKs,
which then activate transcription of genes encoding DNA
synthesis enzymes and S phase cyclin-CDKs. S phase cyclin-
CDKs initially are held in check by inhibitors until G1/S phase
cyclin-CDKs phosphorylate the inhibitors. This triggers their
polyubiquitination by SCF ubiquitin ligase and degradation by
proteasomes. The released S phase cyclin-CDKs then
phosphorylate regulatory proteins bound to chromosomal
replication origins, promoting initiation of DNA synthesis. The
synthesis of mitotic cyclin-CDKs increases in S and G2 phases.
The activities of these complexes initially are blocked by
phosphorylation of CDK subunits, and then are activated later
by dephosphorylation. Once activated, mitotic cyclin-CDKs
phosphorylate a large number of proteins that control
chromosome condensation, retraction of the nuclear envelop,
formation of the mitotic spindle, and alignment of chromosomes
at the metaphase plate.

8. Subsequently, the anaphase promoting complex (APC/C), another
ubiquitin ligase, polyubiquitinates a protein called securin which
helps hold the sister chromatids of metaphase chromosomes
together. The degradation of securin by proteasomes initiates
anaphase and sister chromatids separate. Later in anaphase,
APC/C polyubiquitinates mitotic cyclins leading to their
degradation. Due to the loss of mitotic cyclin-CDK kinase activity
proteins responsible for chromosomal condensation, etc. are
dephosphorylated. Chromosomes then decondense, and nuclear
membranes are re-synthesized. Cells next move forward into
telophase where cytokinesis occurs, completing the cell cycle. In
the ensuing G1 phase, replication origin regulators are synthesized
and pre-replication complexes assemble at origins. This prepares
cells for another round of DNA synthesis in the next S phase.
Due to degradation of regulatory proteins at the G1/S,
metaphase/anaphase, and anaphase/telophase boundaries, the
passage of cells through the cell cycle is irreversible. The G1/S
transition (“START”) is a major checkpoint after which passage
through the cycle becomes independent of mitogens (e.g., growth
factors).
Mechanism of Cell Cycle Regulation (II)

9. Mechanism of Cell Cycle Regulation (III)

10. APC/C Regulation of Sister Chromatid
Separation
Metaphase chromosomes are held together at centromeres via
ring-like proteins called cohesins (Fig. 18.36b, left). Once
spindle-assembly checkpoint processes have been satisfied (see
below), a protein called Cdc20 triggers sister chromatid
separation (Fig. 19.27 right). Cdc20 activates the APC/C
ubiquitin ligase which polyubiquitinates a protein called securin
which is an inhibitor of the enzyme called separase. Once
securin is degraded by proteasomes, separase cleaves the Scc1
component of cohesins resulting in their disassembly and
separation of sister chromatids to the spindle poles.

11. Regulation of Initiation of DNA
Replication by S phase Cyclin-CDKs (I)
In eukaryotic cells, DNA
synthesis occurs
simultaneously at multiple
replication origins which
initiate DNA synthesis
only once per cell cycle.
This ensures that the
number of chromosomes
per cell is correctly
maintained. At the end of
M phase when all M phase
cyclins are degraded, the
dephosphorylated forms of
MCM helicases and two
initiation factors assemble
along with the ORC
(origin recognition complex) at replication origins (Step 1, Fig.
19.19). Then when S phase cyclin-CDKs are activated at the end
of G1, S phase cyclin-CDKs and the DDK kinase phosphorylate
MCM helicases and the two initiation factors (Step 2).
Phosphorylation causes ORC and the two factors to disassemble.

12. Regulation of Initiation of DNA
Replication by S phase Cyclin-CDKs (II)
S-phase cyclin-CDKs also
phosphorylate MCM
helicase activators (red)
(Step 2). Subsequently,
origins are unwound by
active MCM helicases,
DNA polymerases load
onto the origins, and
bidirectional DNA
synthesis ensues (Step
3). The phosphorylated
forms of initiation
factors cannot rebind
DNA at origins, and they
are degraded by the SCF
proteasome. Only after S
phase and mitotic cyclin-CDKs are degraded at the end of
mitosis can the initiation factors be synthesized and accumulate
in their dephosphorylated states, and then assemble again at
replication origins. This ensures that DNA replication occurs
only once per cell cycle.

13. Mitogen-stimulated Gene Expression in
G0-arrested Mammalian Cells
Cells in G0 do not synthesize cyclins or CDKs. The transition
of quiescent cells from G0 to G1 and resumption of the cell
cycle is triggered by growth factors in serum (mitogens).
Shortly after binding to receptors, growth factors turn on
the transcription of early response genes using TFs that
preexist in the cell. Among the early response genes are c-
fos, c-jun, and c-myc These genes turn on the transcription
of delayed-response genes. Included within the latter are the
G1 cyclin-CDKs and a TF called E2F, which is controlled by
the Rb gene (next slide). The synthesis of G1 cyclin-CDKs
propels the cell into G1. Prior to the START point, the
withdrawal of growth factors leads to rapid degradation of G1
cyclin-CDKs and return to G0. At the restriction point, G1
cyclin-CDKs reach irreversibly high levels and cells are
committed to enter S phase. After the restriction point,
growth factors are no longer needed for completion of the
cycle. One role of TGFß is inhibition of G1 cyclin-CDKs.

14. Rb and the START Point
Rb is the prototype tumor suppressor gene. Inactivation of Rb
leads to tumors of the retina in children. Rb also is inactivated
in many other tumors. In non-proliferating cells, Rb protein
binds to E2F, and the complex activates histone deacetylases
leading to gene silencing (Fig. 19.15b). When the expression of
the G1 cyclin-CDKs (cyclin D-CDK4/6) are turned on by a
mitogen, Rb is phosphorylated and active E2F is released. E2F
activates transcription of genes needed for passage into S
phase, namely genes encoding DNA synthesis enzymes, Cyclins E
& A (G1/S phase cyclins), CDK2, and
itself. Cyclins E/A-CDK2 (G1/S
cyclin-CDKs) also phosphorylate Rb.
This occurs even if the mitogen is
withdrawn and is the key control
allowing the cell to pass through the
restriction point. In S, G2, and
mitosis, S-phase and mitotic cyclin-
CDKs continue to phosphorylate Rb.
Only after degradation of mitotic
cyclins at the end of mitosis is Rb
dephosphorylated. Rb then can
inhibit E2F in early G1 and in G0-
arrested cells.

15. Checkpoints in Cell-cycle Regulation
To minimize mistakes in
cell cycle events and
transmission of damaged
DNA or otherwise
abnormal chromosomes
to daughter cells,
numerous quality control
checkpoints regulate
passage of cells through
the cell cycle. For
example, DNA-damage
checkpoints occur at
several steps (Fig.
19.34). If damage is
detected, the cell cycle
is arrested and the
damage repaired, if
possible. Severe DNA
damage may trigger
apoptosis (Chap. 21).

16. The Spindle-assembly Checkpoint
In nondisjunction, chromosomes segregate in anaphase prior to
attachment of the kinetochores of all sister chromatids to mitotic
spindle fibers. This results in unequal segregation of chromosomes
to daughter cells (below left). In trisomy 21, nondisjunction occurs
95% of the time in meiosis I during gametogenesis in the mother.
To prevent nondisjunction, a regulatory mechanism involving the
Mad2 protein which is known as the spindle-assembly checkpoint
operates just prior to anaphase (Fig. 19.35). Mad2 binds to
kinetochores that have not bound to microtubules of the mitotic
spindle. Kinetochore binding activates Mad2, and it in turn inhibits
the activity of Cdc20 which controls the APC/C ubiquitin ligase (Fig.
19.27). This delays degradation of securin and anaphase. Only
after all kinetochores have bound to the spindle is Mad2
inactivated, releasing Cdc20 to trigger securin degradation.