Module 10 + Lecture 10 Flashcards
(77 cards)
1
Q
MPF
A
MPF is a heterodimer of mitotic cyclin and cyclin dependent kinase (CDK)
Exit out of mitosis requires inactive MPF
- By ubiquitin-mediated degradation of Cyclin B
For interphase to occur again, cytosolic phosphatases reverse phosphorylation of required proteins for cell division
2
Q
what is the golgi protein that is phosphorylated during mitosis?
A
GM130
3
Q
Anaphase and Telophase Events - cyclins and MPF
A
Requires decrease in mitotic cyclins and inactivation of MPF
- Requires phosphatases to dephosphorylate MPF target protein
4
Q
which proteins are phosphorylated during prophase?
- allow chromo condense
A
- Condensins
- Cohesins
- Histones (H1 and H3)
- Topoisomerase
5
Q
DNA is organized around these essential chromosomal binding proteins
A
histones
- 5 types
- H1 and H3 required
6
Q
what is the kinase that phosphorylates histone H1 and H3?
A
Aurora B
7
Q
H1 vs H3
A
H3:
is part of an octamer that forms the protein core around which DNA is wound to create the nucleosome
H1:
Linker in between neighboring nucleosomes
8
Q
What proteins form the cohesin complex? Function?
A
cohesins
- complex needed to hold sister chromatids together after replication until anaphase
9
Q
Phosphorylates cohesin subunits and allows cohesin dissociation along chromosome arms
A
CyclinB-CDK
10
Q
Release of cohesins along chromosome arms allows assembly of _________ protein complex at prophase
A
condensin
11
Q
What phosphorylates condensins? Where?
A
CyclinB-CDK
carboxyl-terminal domain
12
Q
Structure of cohesin vs. condensin complex
A
Cohesin Complex = circular structure which units 2 diff. DNA molecules
Condensin Complex = diamond-shaped structure which associate with different regions of the same DNA molecule to cause condensation
13
Q
Decondensation
A
Occurs after telophase and mitosis exit:
- Condensins and histones become dephosphorylated
- absence of Cyclin B/CDK
- presence of phosphatases
14
Q
Nuclear envelope presence
A
interphase
- intact
prometaphase
- fragmenting
metaphase
- none
- fragmented into small vesicles
15
Q
Nuclear envelope structure
A
Made of 2 lipid bilayers
1. ONM - Outer Nuclear Membrane:
Continuous with rER membrane
2. INM - Inner Nuclear Membrane:
Associated with scaffold of intermediate filaments (nuclear lamina)
pore complexes (NPCs) span both
16
Q
NucEnv during different phases
A
During Interphase:
- Chromosomal proteins interact with the nuclear lamina to anchor chromatin
- regulate gene expression
During Prophase:
- All 3 lamin proteins are phosphorylated at their serine residues by Cyclin B/CDK
- Initiates disassembly of nuclear lamina = frag. of NE
17
Q
This lamin stays associated with the nuclear membrane during fragmentation
A
lamin B
Lamin A and Lamin C dimers are soluble and go into the cytosol
18
Q
Is lamin A is necessary for nuclear lamina disassembly and nuclear envelope breakdown
A
YES
19
Q
What shape does lamin make
A
tetramers that formed intermediate filaments
#
20
Q
What dephosphorylates lamin - makes it active?
A
Telophase:
Inactivation of Cyclin B/CDK and phosphate Cdc14
21
Q
Karyomere Fusion + mediated by
A
- Vesicles accumulate around an individual chromosome, mediated by chromosomal proteins
- Vesicles begin to coalesce to form a membrane around SINGLE chromosomes - karyomere
- Karyomeres of individual chromosomes fuse together to form the nuclear envelope
Mediated by Rab-like proteins
22
Q
Dictyokinesis
A
golgi division
Organelles must be distributed fairly between the 2 daughter cells
- Before cytokinesis, vesicles and tubules of the fragmented GA accumulate in 2 clusters - one at each spindle pole
23
Q
Fragmentation requires phosphorylation of at least ________
A
1 Golgi protein
- GM130 by Cyclin B/CDK in mitosis
@ serine 25 - PS25
24
Q
G0 in mammal cells
A
- In mammals, most of the body’s cells have entered G0 and will never re-enter
- Other cells withdraw temporarily from the cell cycle and will re-enter G1 when receiving the appropriate signal
25
Yeast vs. Vertebrate Cyclin Requirements
Yeast requires a single cyclin and single CDK for regulation
Vertebrates require multiple Cyclin and CDK variants:
Early G1: D-type cyclins: CDK4 or CDK 6
Triggers S-Phase: Cyclin E-CDK2
Completes S-Phase: Cyclin A-CDK2
Mitosis: CDK1-Cyclin B
26
Regulating entry into G1
- requires expression of D-type cyclins and CDK4/6
- Mitogens or growth factors are signaling molecules that induce cell division by expressing the G1 Cyclin/CDK
27
The Restriction Point
an important regulatory checkpoint in the cell cycle becomes independent of the presence of the mitogen
Passage through the “restriction point” correlated with end of Cyclin D/CDK4-6 activity and beginning of Cyclin E/CDK2 expression
28
What changes in gene expression allow a cell paused at G0 to continue into the cell cycle?
- Early response genes (peak at 1 hr)
- Late response genes (begins at 2 hrs)
29
Why are there 2 responses? (early and late)
Early response gene expression is regulated by transcription factors that are already present in the cell (need to be activated by phosphorylation)
- Includes SRF and TCF transcription factors
- Activated by MAP-kinase (endpoint of receptor tyrosine kinase) signal transduction pathway
30
When cells enter cell cycle from G0
EGF - Epidermal Growth Factor
Transcription Factors: c-Fos and c-Jun , E2F
which code for…
- Cyclin D, Cyclin E
- Cdk2, Cdk4, Cdk6
Cdk 4 and Cdk 6 are D-type cyclins are expressed first
Then Cyclin E and Cdk2
31
Early gene expression + example
- regulated by MAP-kinase
- Culmination of tyrosine kinase receptor activation
- Activated by SRF (serum response factor) and TCF (ternary complex factor) transcription factor
c-Fos transcription factor will activate delayed gene expression
32
Impact of turning off early response genes
- When mitogens are added, protein synthesis inhibitors are also added
= no delayed response, yes early response (stays high)
No early response proteins, no E-R transcription factors to transcribe delayed response genes.
= No ER proteins to down-regulate ER genes
33
Removing Mitogen before vs after the restriction point
Removing Mitogen BEFORE the restriction point:
Decreases CYclin D/CDK 4-6 levels
Cell does not proceed into S-phase
Cells go back into G0 (quiescent cells)
Removing Mitogen AFTER the restriction point:
Cyclin E/CDK 2 expression is high
Decrease in Cyclin D does not affect progression into S-phase
Leads to completion of cell division (Dividing cells)
34
Biochemical explanation of restriction point
- Necessary components of the restriction point are delayed response proteins
- E2F is a transcription factor the regulated expression of genes required in S-phase
- E2F is inactivated by association with inhibitory protein (ex. Rb)
Inactivation = Cell Cycle PROGRESSION
35
E2F positive feedback loop
Active E2F induces expression fo Cyclin E/CDK2:
- Targets Rb for phosphorylation
- When enough E2F is activated it maintains active state by:
- Inducing expression for Cyclin E
- E2F gene
Therefore this is a POSITIVE feedback loop
36
C2 types of DNA damage
- Ionizing radiation creates double-stranded DNA breaks (ATM)
- UV radiation creates thymine nucloetide dimers (ATR)
37
ATR
- Kinase, activated when bound to thymine dimers in the affected DNA molecule
- Target of ATR is Chk1
- Chk1 is also a kinase and its target is Cdc25
- Cdc25 is a regulator of CDK activity and MPF activity at G2
→ M phase transition
38
Long‐term effect of Cdc25 phosphorylation
sequestered in the cytosol through association with a cytosol adaptor protein, 14‐3‐3
39
ATM Kinase
recognizes double-stranded breaks in DNA
Binding to substrate activates ATM
Target of ATM is Ctk2 kinase
Phosphorylated Chk2 is active and targets the p53 protein
P53 is an unstable protein and after translation it is immediately ubiquitinylated and degraded
But phosphorylated p53 is stable and is a transcription factor tat activated transcription of target genes:
P21/CIP - a general Cyclin/CDK INHIBITOR
P21 will bind to G1 Cyclin in the case of a double-stranded break and stall G1
After repair, ATM dissociates and allows progression of cell cycle
40
Constant activation of p53 (for a long time)
will induce expression of pro-apoptotic genes can cause cell death
41
Spindle Assembly Checkpoint - SAC
Cell cycle will delay in metaphase if even one chromosome/sister chromatid is not attached to the mitotic spindle
Checkpoint detects:
The association of the kinetochore proteins to microtubules of mitotic spindle
42
Nondisjunction in SAC mutant
- Cell proceeds into anaphase even though 1 chromosome is not attached to the mitotic spindle
- The chromosome is not pulled apart to opposite cells so would stay on one side
- Results in nondisjunction and aneuploidy
43
Mad2
Mad2 inactivates APC-Cdc20
Causes securin protein to remain intact and prevents activation of Separase - so sister chromatid separation cannot occur
→ If there is a loss of function mutation in Mad2, it causes the cell to proceed into anaphase prematurely.
44
Mad2 conformations
open and closed
Mad 2 and Mad 1 associate on the chromosome, making Mad 2 go from open to closed conformation
When Cdc20 in open conformation interact with CDC20 in closed conformation, they all turn to closed conformation (similar to prions)
45
Passing the SAC
When:
Every replicated chromosome has bipolar attachment to the mitotic spindle
Both kinetochores are attached to microtubules
… Mad 1 and Mad 2 proteins are released from the kinetochore
46
Mad2, Cdc20 affinity, and APC
Binding of p31 to closed conformation of Mad 2 leads complex away from chromosomes and changes Mad2 to open state
- In open state, Mad 2 has less affinity for Cdc20
- Causes inactivation of checkpoint
APC-Cdc20 is activated:
- Targets Securin for degradation and releases inhibition on Separase
- Allows anaphase to occur
47
Events associated with exit from mitosis
- Reassembly of nuclear envelope
- Chromosome decondensation
- Reassembly of mitochondrial, Golgi and ER networks
- Disassembly of mitotic spindle
48
Requirements to exit mitosis
INACTIVATION of MPF
- Requires activity of APC-Cdh1 which targets Cyclin B for degradation
49
Role of MEN; Mitotic Exit Network checkpoint
To monitor completion of anaphase and activate APC-Cdh1
- Cdh1 needs to be dephosphorylated to be bind to APC, which requires Cdc14
key feature; Cdc14
50
Studying the MEN with yeast model system
During interphase and early mitosis - Cdc14 is located in the nucleolus which is associated with rRNA synthesis (Cdc14 is inactive)
When anaphase occurs, Cdc14 is released and activated
51
small monomeric GTPase that is associated with the spindle pole body closest to the daughter bud
Tem1
52
GTPase accelerating protein that keeps Tem1 in the GDP bound form
Tem1-GAP
53
associated with the membrane of the daughter cell and is not near the Tem1 protein while the cell is in metaphase
Tem1-GEF
54
what is another target of Cdc14, other than Cdh1?
Sic1
55
How tumor cells differ from normal cells (3 ways)
- point mutations, gene deletions, chromosome gain or loss
- made of multiple cells types and grow maximally
- migratory pathways and secondary sites of growth
56
Cell changes that cause cancer (6)
- resist cell death
- sustain proliferative signalling
- evade growth supressors
- induce angiogenesis
- enable replicative immortality
- activate invasion and metastisis
57
Energy producton in cancer cells
- tumors rely on glucose = sequestered in their cells
- produce very little ATP/mol glucose
- PET/CT scans can then detect cancer
58
Cancer cell contact-inhibition
normal cells
- contact-inhibited growth
- align in an orderly fashion
cancerous
- NOT contact-inhibited
- covered with hairlike stuff and bulbous projections
- rounded cells
59
Metastasis
Metastatic cancer cellls use invadopodium to penetrate basement membranes and migrate to distant sites in the body
60
Developing cancer
- Carcinogens (direct and indirect) mutate DNA
- Multi-hit model: multiple mutations required to cause cancer
- Mutations in tumor-suppressor genes and proto-oncogenes (colon cancers)
- Oncogenes work synergistically
(active oncogenes = cancer)
61
Benzo(a)pyrene carcinogen
- in cigarette smoke and coal tar
- P-450 genes modify to produce a potent mutagenic species that reacts with DNA
62
Cancer multi-hit model
cancers arise by a clonal selection process
First mutation:
- gives one cell a slight growth advantage
Second mutation:
- in a progeny cell causes its descendants to grow more uncontrollably and form a small benign tumor
Third mutation:
- in a cell with the tumor allows it to overcome constraints imposed by the tumor microenvironment
Fourth mutation:
- in one cell allows its progeny to escape into the bloodstream and establish daughter colonies at other sites (metastatic)
63
Does the multi-hit model of cancer predict increase in cancer incidence with age
yes
accumulate mutations
64
Sunburn and cancer
UV exposure causes p53 (tumor suppressor gene) mutations
follows multiple-genetic hit model
1. normal tissue contains a few cells that have driver mutations
2. clones of cells with p53 mutations are found in tissue
65
Mouse mammary tumor virus
- MMTV mammary tumor breast-specific promoter drives Myc and Ras oncogenic forms
- mice with both Myc and Ras are much-more tumor prone
= synergestic effect of multiple mutations
66
Metastasis of human colorectal cancer
multi-hit model
- APC tumor-suppressor gene mutation in a single epithelial cell
- causes localized benign polyp
- expression of Ras oncoprotein and loss of tumor-suppressor p53 gene
- additional mutations permit metastasis (invade basement and blood vessels)
67
Genetic basis of cancer
Gain-of-function mutations in oncogenes (dominant)
Loss-of-function mutations in tumor-suppressor genes (recessive)
Loss-of-function mutations in genome maintenance genes (recessive)
- propensity for developing cancer is heritable
68
What do tumor-supressor genes encode
- proteins that directly or indirectly control cell-cycle progression
Inhibit cell survival or proliferation
68
What do proto-oncogenes encode
- growth-promoting signals/receptors
- signal-transducing proteins
- transcription factors
- anti-apoptotic proteins
promote cell survival or proliferation
69
What do genome maintenance genes encode
- DNA repair enzymes
Repair or prevent DNA damage
70
7 types of mutant proteins that lead to cancer
1. extracellular signalling molecules
2. signal receptors
3. signal-transducing proteins
4. transcription factors
5. cell-cycle control proteins
6. DNA-repair proteins
7. apoptotic proteins
71
Retinoblastoma: spontaneous somatic mutation (2 types)
Both alleles of a tumor-suppressor gene must be lost (recessive)
loss of hetero = bad
a) Hereditary
- got one bad gene from parent
- loss of function of other Rb allele
b) Sporadic
- happen to lose function in both
- 2 loss of function mutations
72
2 mechanisms for loss of hetero of tumor-suppressor genes
a) Mis-segregation
- split 1:3
- 3 = abb
- either becomes ab OR bb
bb is bad bc homo
b) Mitotic recombination
- during recombination they both happen to get the bad allele
- homo for mutant allele
73
Chronic myelogenous leukemia (CML)
- Pjiladelphia chromosome
- due to a translocation between ends of chromosomes 22 and 9
- forms hybrid gene encoding chimeric BCR-ABL protein
treatment:
gleevac/imatinib binds to BCR-ABL active site and inhibits its kinase activity
74
Misregulation of cell growth and death in cancer
- tumor cells produce constitutively active forms of intracell signal proteins
- inappropriate production of gene transcription factors can induce cell transformation
- loss of growth regulators or apoptotic proteins can lead to cancer
75
Proto-oncogenic mutations
- result in overproduction or unregulated constitutive activity of proteins that promote cell proliferation and transformation
treatment:
- gefitini/iresszza inhibits kinase activity
- trastuzumab can target HER2 on breast cancer
76
Burkitt's lymphoma
- translocation
