Cell-Cycle Cell-Cycle Regulation and the Regulation and the Genetics of CancerGenetics of Cancer
OutlineOutline
The control of cell divisionThe control of cell division Normal cell cycleNormal cell cycle Yeast as a model organismYeast as a model organism Cell cycle control: Molecular MechanismsCell cycle control: Molecular Mechanisms Regulation of cyclin-CDK activity Regulation of cyclin-CDK activity Checkpoints that regulate passage Checkpoints that regulate passage
through cell cyclethrough cell cycle
The normal cell divisionThe normal cell division
Cyclin-dependent kinases Cyclin-dependent kinases (CDKs) collaborate with (CDKs) collaborate with cyclins to ensure the proper cyclins to ensure the proper timing and sequence of timing and sequence of cell-cycle eventscell-cycle events
Experiments with yeast helped Experiments with yeast helped identify genes that control cell identify genes that control cell
divisiondivision Properties of yeast Properties of yeast
Grow as haploid or diploid organismsGrow as haploid or diploid organisms Can identify recessive mutations in haploidsCan identify recessive mutations in haploids Complementation analysis in diploidsComplementation analysis in diploids
Budding – daughter cell arises on surface Budding – daughter cell arises on surface of mother cell and grows in size during of mother cell and grows in size during cell cycle. Helps determine stage of cell cell cycle. Helps determine stage of cell cycle.cycle.
Isolation of temperature-Isolation of temperature-sensitive mutants in yeastsensitive mutants in yeast
Mutants grow Mutants grow normally at normally at permissive permissive temperaturetemperature
Mutants loses gene Mutants loses gene function at function at restrictive restrictive temperaturetemperature
Thousands of cell Thousands of cell cycle mutants have cycle mutants have been identifiedbeen identified
A cell-cycle mutant in yeastA cell-cycle mutant in yeast (a) growth at (a) growth at
permissive permissive temperature displays temperature displays buds of all sizesbuds of all sizes
(b) growth at (b) growth at restrictive restrictive temperature shows temperature shows cells have finished cells have finished first cell cycle and first cell cycle and arrested in the secondarrested in the second
A double mutant reveals A double mutant reveals which mutation is neededwhich mutation is needed
Human CDKs and cyclins can function in yeast in place of native proteins
70 cell-cycle genes identified 70 cell-cycle genes identified through temperature-sensitive through temperature-sensitive
mutation screensmutation screens
Cell Cycle Control: Cell Cycle Control: Cyclin-dependent kinases (CDK)Cyclin-dependent kinases (CDK) and their
regulatory subunits, the cyclins
CyclinsCyclins
•50-90kDa proteins with conserved ‘cyclin box’ region
•Forms 5 alpha helices.
•Conservation between Rb and TFIIB suggests that cyclin box domain regulates protein interactions related to cdk regulation and transcription
Cyclin – CDK interactionsCyclin – CDK interactions
Cdks ( blue) by themselves are inactive. Activation occurs through phosphorylation of the T loop (green) and the binding of cyclin (purple) at the PSTAIRE helix (red). These events lead to a conformational change that produces a functional active site (yellow)
How enzymes select their substrateHow enzymes select their substrate
a, b, In general, enzymes recognize their targets through structural complementarity between the substrate and the enzyme's active site (indicated here by the shape of the 'pocket'). Small substrates (a) and relatively small modification sites on proteins (b) can be recognized by this mechanism.
c, Some enzymes make additional, specific contacts with the substrate that enable them to distinguish between proteins that have identical or related sites of modification.
d, cyclin-dependent protein kinases (CDKs) have relegated that function to the exchangeable cyclin subunit, enabling a single CDK catalytic subunit to exist in numerous forms with different specificities.
Cell cycle: cyclin guides the way by C WittenbergNature 434, 34-35 (3 March 2005)
Higher eukaryotes have more forms of both cyclins and CDKs compared to lower
eukaryotes.
CDK1
Cyclin-dependent kinases (CDKs) control the Cyclin-dependent kinases (CDKs) control the cell cycle by phosphorylating cell cycle by phosphorylating specific serine
and threonine residues of select proteins during different phases of the cell cycle
other proteinsother proteins
E.g. E.g. Nuclear laminsNuclear lamins
CDK substratesCDK substrates Underlie inner surface of the nuclear membraneUnderlie inner surface of the nuclear membrane Probably provide structural support for nucleusProbably provide structural support for nucleus May also be site for assembly of DNA replication, May also be site for assembly of DNA replication,
transcription, RNA transport, and chromosome structure transcription, RNA transport, and chromosome structure proteinsproteins
Dissolution of nuclear membrane during mitosis is triggered Dissolution of nuclear membrane during mitosis is triggered by CDK phosphorylation of nuclear laminsby CDK phosphorylation of nuclear lamins
REGULATON OF CYCLIN–CDK ACTIVITY REGULATON OF CYCLIN–CDK ACTIVITY
1. 1. Cyclin availabilityCyclin availability
Association with a cyclin is absolutely required for Association with a cyclin is absolutely required for Cdk activity.Cdk activity.
Cyclin levels can be changed by transcriptional Cyclin levels can be changed by transcriptional regulation and/or by ubiquitin-dependent regulation and/or by ubiquitin-dependent proteolysis. proteolysis.
E.g. cyclins D and E contain a PEST sequence E.g. cyclins D and E contain a PEST sequence [segment rich in proline(P), glutamic acid (E), [segment rich in proline(P), glutamic acid (E), serine (S) and threonine (T) residues]: which are serine (S) and threonine (T) residues]: which are required for efficient ubiquitin-mediated cyclin required for efficient ubiquitin-mediated cyclin proteolysis at the end of a cell cycleproteolysis at the end of a cell cycle
REGULATON OF CYCLIN–CDK ACTIVITY REGULATON OF CYCLIN–CDK ACTIVITY 2. 2. Inhibitory phosphorylationInhibitory phosphorylationCyclin–Cdk complexes can also be inactivated by Cyclin–Cdk complexes can also be inactivated by
phosphorylation of tyrosine and threonine phosphorylation of tyrosine and threonine residues close to the active site of the Cdk residues close to the active site of the Cdk subunit. subunit.
This phosphorylation is mediated by Wee1-type This phosphorylation is mediated by Wee1-type protein kinases, and the inhibitory phosphate protein kinases, and the inhibitory phosphate groups are removed by Cdc25-type groups are removed by Cdc25-type phosphatasesphosphatases
REGULATON OF CYCLIN–CDK ACTIVITY REGULATON OF CYCLIN–CDK ACTIVITY 3. 3. Stoichiometric inhibitionStoichiometric inhibition
CDK activity can be regulated by stoichiometric CDK activity can be regulated by stoichiometric inhibitors (cyclin kinase inhibitorsinhibitors (cyclin kinase inhibitors--CKIsCKIs), which ), which bind to CDK alone or to the CDK-cyclin complex bind to CDK alone or to the CDK-cyclin complex and regulate CDK activity. and regulate CDK activity.
Lower eukaryotes possess a single CycB–Cdk1 Lower eukaryotes possess a single CycB–Cdk1 specific inhibitor, whereas in higher eukaryotes specific inhibitor, whereas in higher eukaryotes 2 distinct families exist; the INK4 family and 2 distinct families exist; the INK4 family and Cip/Kip familyCip/Kip family
REGULATON OF CYCLIN–CDK ACTIVITY REGULATON OF CYCLIN–CDK ACTIVITY
3. 3. Stoichiometric inhibition…Stoichiometric inhibition…
CKIs are regulated both by internal and external CKIs are regulated both by internal and external signals: signals:
E.g. expression of p21 is under transcriptional control E.g. expression of p21 is under transcriptional control of the p53 tumour suppressor gene (internal), of the p53 tumour suppressor gene (internal), whereas the expression and activation of p15 and whereas the expression and activation of p15 and p27 increases in response to transforming growth p27 increases in response to transforming growth factor b (TGF-b), contributing to growth arrest factor b (TGF-b), contributing to growth arrest (external)(external)
REGULATON OF CYCLIN–CDK ACTIVITY REGULATON OF CYCLIN–CDK ACTIVITY 4. 4. Intracellular localisationIntracellular localisation
Intracellular localization of different cell cycle-Intracellular localization of different cell cycle-regulating proteins also contributes to CDK regulating proteins also contributes to CDK regulationregulation
Checkpoints integrate repair of chromosome damage with events of cell cycle
G1-S checkpoint p53 – transcription factor
that induces expression of DNA repair genes and CDK inhibitor p21
p53 pathway activated by ionizing radiation or UV light (causing DNA damage) during G1 phase delays entry into S phase
DNA is repaired before cell cycle continues
If DNA is badly damaged cells commit suicide (programmed cell death or apoptosis)
G1-S phase transition
Mutations in p53 disrupt G1-S transition
Gene amplification in tumour cells that appear as homogenously staining regions (HSR)
Small chromosome-like bodies (called minutes) in tumour cells that lack centromeres and telomeres
p53 mutants do not induce p21 and cell cycle is not arrested
Cells replicate damaged DNA
Cells die or DNA is degraded and cell is engulfed and digested by neighboring cells (apoptosis, or programmed cell death)
S phase checkpoint
Individuals affected by ataxia telangiectasia (AT), an autosomal recessive disorder, are unable to slow down DNA replication after exposure to radiation. The AT gene (ATM) codes for a protein kinase with homology to the catalytic domain of phosphatidylinositol 3-kinase (PI-3 kinase) and serves as a checkpoint gene in response to DNA damage. ATM is central to dsb responses.
G2-M transition is controlled by phosphorylation and dephosphorylation
Two checkpoints act at the G2-M transition
double strand breaks
Checkpoint in MCheckpoint in Mspindle damagespindle damage
Checkpoints ensure genomic stability
Defective checkpoints Chromosome aberrations
Aneuploidy Changes in ploidy
Single-stranded nicks – normally repaired in G1 phase
Chromosome loss or gain – normally corrected in G2-M checkpoint
Three classes of error lead to aneuploidy in tumor cells
Normal cells Cancerous cells
General reading: MBoC by Alberts et al (4th ed): pgs 863-906 OR Cancer Biology by RJB King : pgs 148 - 158……..ORChapter 9 Mol & Cell Biol of Cancer by Knowles and Selby
Optional reading: The cell cycle: a review…. targets in cancer by K Vermeulen, DR. Van Bockstaele and ZN. Berneman Cell Proliferation (June 2003) 36(3) pp131-149.
Cell cycle by Gary S Stein et al www.els.net