Download Biochemical Control of the Cell Cycle

Biochemical Control of the
Cell Cycle
BNS230
Lecture programme
• Three lectures
• Aims
– Describe the cell cycle
– Discuss the importance of the cell cycle
– Discuss how the cycle is regulated
Cell division
15 hours
M
phase
G0 state
G2 phase
G1 phase
12 hours
S-phase
(DNA synthesis
5 hours
16 hour cell cycle
Cell cycle definition
• A series of distinct biochemical and
physiological events occurring during
replication of a cell
• Occurs in eukaryotes
• Does not occur in prokaryotes
• Time of cell cycle is variable
Cell cycle timing
•
•
•
•
Yeast 120 minutes (rich medium)
Insect embryos 15-30 minutes
Plant and mammals 15-20 hours
Some adults don’t divide
– Terminally differentiated
– e.g. Nerve cells, eye lens
• Some quiescent unless activated
– Fibroblasts in wound healing
Components of the cell cycle
• M phase
– Cell division
– Divided into six phases
•
•
•
•
•
•
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
Cytokinesis
Components of the cell cycle
• G1 phase
– Cell checks everything OK for DNA
replication
– Accumulates signals that activate
replication
– Chloroplast and mitochondria division not
linked to cell cycle
Components of the cell cycle
• S-phase
– The chromosomes replicate
– Two daughter chromosomes are called
chromatids
– Joined at centromere
– Number of chromosomes in diploid is four
Components of the cell cycle
• G2-phase
– Cell checks everything is OK for cell
division
– Accumulates proteins that activate cell
division
Why have a cell cycle?
• Comprises gaps and distinct phases of
DNA replication and cell division
• If replicating DNA is forced to condense
(as in mitosis) they fragment
• Similarly if replication before mitosis
– Unequal genetic seperation
• I.e. Important to keep DNA replication
and mitosis separate
Why have a cell cycle?
• Important to have divisions in mitosis
• e.g. Important metaphase complete
before anaphase. Why?
• If not segregation of chromosomes
before attachment of chromatids to
microtubles in opposite poles is possible
• Down syndrome due to extra
chromosome 21
Why have a cell cycle?
• Gaps provide cell with chance to assess
its status prior to DNA replication or cell
division
• During the cell cycle there are several
checks to monitor status
• These are called checkpoints
Checkpoints
• Checkpoint if G1 monitors size of cell in
budding yeast (Saccharomyces
cerevisae)
• At certain size cell becomes committed
to DNA replication
• Called start or replication site
Evidence of size checkpoint
• Yeast cells (budding yeast) grown in
rich medium
• Switch to minimal medium
• Cells recently entering G1 (buds)
delayed in G1 (longer to enter S-phase)
• Large cells above threshold size still go
to S-phase at same time as in rich
medium
Evidence of size checkpoint
• Yeast in rich medium
– 120 minute cell cycle
• Short G1 phase
• Yeast in minimal medium
– Eight hour cell cycle primarily because of
long G1 phase
Checkpoints
• Checkpoint 2 in G1 monitors DNA
damage
• Evidence?
– Expose cells to mutagen or irradiation
– Cell cycle arrest in either G1 phase or G2
phase
• The protein p53 involved in cell cycle
arrest
– Tumour suppresser
Checkpoints
• Checkpoint in S-phase monitors
completion of DNA replication
– Cell does not enter M-phase until DNA
synthesis is complete
• Checkpoint in G2
– DNA breaks cause arrest
– Otherwise when chromosomes segregate
in mitosis DNA distal to breaak won’t
segregate
Checkpoints
• Checkpoint in mitosis
– Senses when mitotic spindles have not
formed
– Arrests in M-phase
– Otherwise unequal segregation of
chromosomes into daughter cells
• Described cell cycle, now I will talk
about genes and proteins that control
this process
Molecular control of cell cycle
• Two experimental approaches
– Biochemical
•
•
•
•
Sea urchin fertilised eggs
Rapid
Synchronous division
Analyse proteins at various stages of cycle
– Genetic analysis using
• Budding yeast Saccharomyces cerevisae
• Fission yeast Schizosaccharomyces pombe
Using genetics to study the cell cycle
• To study the genetic basis of a
biological event
– Make mutants defective in that event
– Determine which genes have been
mutated
– Understand role of gene (and encoded
protein) in the event
– Problem: How do you make mutants that
disrupt the cell cycle
– Cells will not replicate
Using genetics to study the
cell cycle
• Isolate temperature sensitive mutants
that have defect in cell cycle
• At low temperature these mutants
progress through cell cycle
• Arrest in cell cycle at elevated
temperature
• Mutation causes gene product (protein)
to be highly sensitive to temperature
Using genetics to study the
cell cycle
• Isolation of genes that regulate the cell
cycle
• Step 1: Create strains with mutations in
cell cycle genes
Isolating cell cycle mutants
Yeast culture
(S. pombe)
Mutagenise and plate
out at high and low temperature
37°C
30°C
Colonies 4 and 10 are possible cell cycle mutants. Called
cell division cycle (cdc) mutants >70 cdc mutants isolated
Are the temperature sensitive mutants
cdc mutants?
Grow colonies at 30°C
Shift temperature to 37°C
Look under a microscope
Colony 4: Too small; enters mitosis too
early (Wee 1 mutant)
Colony 10: very long stuck in G2
(cdc25 mutant)
Wild type cells
Using genetics to study the
cell cycle
• Step 2: Insert plasmids containing
fragments of wild type DNA
• Step 3: Look for plasmid that corrects
genetic defects
• Step 4: Plasmid contains a cell cycle
control gene
What do we do with the mutants?
Use mutants to isolate cdc genes and
then study what the proteins do
Wild
type
S. pombe
Extract DNA
Wee1
Yeast vector
cdc25
Cut with restriction enzyme
and ligate into vector
Take recombinant vectors and
transform into cdc mutants
• Wee mutant with normal gene wee1
gene in plasmid will grow at 37
• cdc25 mutant with normal cdc25 gene
in plasmid will grow at 37
• I.e gene in recombinant plasmid is
complementing the mutation
Biochemical studies
• 1st evidence proteins regulate cell cycle
– Fuse interphase cells (G1, S or G2) withMphase cells
– Cell membranes breakdown and
chromosomes condense
– I.e Mitotic cells produce proteins that cause
mitotic changes in other cells
Microinjection with frog oocyte
• Oocyte stays in G2-phase
• Male gets busy and female produces
progesterone
• Oocyte enters mitosis
• Purify proteins from oocyte cells treated
with progesterone
• Inject into G2 arrested cells and see
which protein causes mitosis (1971)
MPF
• Protein identified that causes mitosis
• Called maturation promoting factor
• MPF in all mitotic cells from yeast to
humans
• Renamed mitosis-promoting factor
Properties of MPF
• MPF activity changes through the cell cycle
• MPF activity appears at the G2/M interphase
• and then rapidly decrease
How does MPF cause mitosis?
• It’s a protein kinase
– Phosphorylates proteins
• Phosphorylates proteins involved in
mitosis
• Phosphorylates histones causing
chromatin condensation
• Phosphorylates nuclear membrane
proteins (lamins) causing membrane
disruption
Characterisation of MPF
• Consists of two subunits; A and B
• Subunit A: Protein kinase
• Subunit B: Regulatory polypeptide
called cyclin B
• Protein kinase present throughout cell
cycle
• Cyclin B gradually increases during
interphase (G1, S, G2)
• Cyclin B falls abruptly in anaphase
(mid-mitosis)
What does this profile tell you?
MPF not just due to association of subunits A and B
other factors involved
Protein kinase (subunit A)
Cyclin B levels (subunit B)
MPF activity
G1
S
G2
M
MPF}
Cyclin B (subunit B)
Protein kinase (subunit A)
Metaphase
Ubiquitin
Anaphase
Prophase
Interphase
(G1-S-G2)
Telephase
Proteosome
Cyclin B
• How do Cyclin B levels decrease
abruptly
• Proteolytic degradation
• Degraded in a protease complex
present in eukaryotic cells called “The
Proteosome”
• Specific proteins degraded by complex
when tagged by a small peptide called
ubiquitin
Cyclin B
• Cyclin B is tagged for Proteosome
degradation at anaphase
– Tagged at N-terminus at sequence called
– Destruction box
– DBRP binds to Destruction box
• Guides Ubiquitin ligase to add ubiquitin
molecules to Cyclin B
• Why is Cyclin B only degraded in
anaphase
Destruction
box
P
P
DBRP
(active)
Protein
de-phosphorylase
MPF?
DBRP
(inactive)
Ubiquitin ligase adds ubiquitin
when DBRP binds to the
destruction box
DBRP = Destruction box recognition protein
Cyclin B
• DBRP is normally inactive and is only
activated in anaphase via phosphorylation
• Possible MPF phosphorylates DBRP causing
Cyclin B destruction
– Binds to the destruction box
– Activates ubiquitin ligase to add ubiquitin to
Cyclin B
– Cyclin B then targeted to the Proteosome for
degradation
Cyclin B
• When this causes MPF inactivation
– DBRP dephosphorylated by constitutive
phosphorylase
• Other proteins also control MPF
– Activity doesn’t increase as Cyclin B increases
• Proteins discovered in yeast by cdc mutant
complementation
inactive
Cyclin B (subunit B)
Protein kinase (subunit A)
cdc13
cdc2
MPF}
Y15 T161
Wee1
P Y15
CAK
Inactive MPF
T161
Inactive MPF
P Y15
cdc25
T161
Y15 T161
P
P
Active MPF
MPF activity
• Wee mutant small: Enters mitosis
prematurely
• cdc 25 mutant long: Stays in G2 for
longer
• Wee phosphorylates Y15 and
inactivates MPF
• CAK (cdc2 [MPF]-activating kinase)
phosphorylates T161
• cdc25 dephosphorylates Y15 and
activates MPF
Cell cycle
• How is entry into S-phase controlled?
• Throughout cell cycle the protein kinase
(cdc28 in sc and cdc2 in sp) binds to
specific cyclins
• This changes the specificity of the
protein kinase
Activity of Protein Kinase
• Cdc28-cyclins B1-4: Protein kinase
activates proteins involved in early
mitosis by phorphorylating them
• Cdc28-cyclins 1-3: Protein kinase
activates proteins involved in initiation of
DNA replication by phosphorylating
them
• cdc28-cyclin 5: Phorphorylates and thus
activates proteins that maintain DNA
replication
How many protein kinases?
• In both yeasts only one protein kinase
• In higher eukaryotes multiple protein
kinases
– Active at different stages of the cell cycle
• As with yeast different cyclins