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287 Cards in this Set
- Front
- Back
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How does GPCR activation occur?
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- binding of a hormone induces a conformational change in intracellular domain
- G-protein moves laterally through bi-lipid and binds to receptor |
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What happens when G-protein binds to GPCR?
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- conformational change in alpha subunit leading to guanine exchange
- GDP is released, GTP comes in due to high [GTP] in cytosol - alpha subunit disassociates from beta-gamma complex |
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How does the effector in GPCR pathway get activated?
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- hormone dissociates from receptor
- alpha subunit binds and activates effector - GTPase (fast) hydrolyzes GTP to GDP - alpha subunit goes back to beta-gamma complex |
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What are B-adrenergic receptors?
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- coupled to stimulatory G-proteins (Gs) with 3 subunits
- liver & adipose cells: glycolysis and lipolysis - heart muscle: increase contraction, increase blood supply to tissues - SM cells of intestine: increase relaxation, save energy for major locomotary muscles |
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What do G-proteins associated with B-adrenergic receptors do?
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- Gsa-GTP leaves Gby complex
- binds and activates adenylyl cyclase - cAMP is increased - intrinsic Gsa-GTPase hydrolyzes GTP to GDP - Gsa-GDP attaches back to Gby complex - adenylyl cyclase is deactivated and cAMP decreases |
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What are a2-adrenergic receptors?
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- have same Gby complex as beta receptors but Gia subunit
- blood vessels of SM of intestine, skin, kidney: arteries constrict, blood supply reduced to periphery --> INHIBITORY |
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What do G-proteins associated with a2-adrenergic receptors do?
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- interacts with different region of adenylyl cyclase catalytic domain
- inhibits adenylyl cyclase - net decrease in cAMP |
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When are a2-adrenergic receptors activated?
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- in response to adenosine and prostaglandin (PGE1)
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How are levels of cAMP fine-tuned in cells?
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There are different ligand-receptor complexes that stimulate and inhibit adenylyl cyclase
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What does adenylyl cyclase do?
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It takes ATP and converts it to cAMP by removing two PP and making new bonds
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What's the purpose of adenylyl cyclase in the pathway?
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It uses a little bit of ATP to make even more ATP for cells through amplification
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What happens when adenylyl cyclase is turned off?
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- cAMP degradation by phosphodiesterases
- cAMP --> 5' AMP - cAMP is unstable and phosphodiesterase is always active and non-regulated so cAMP is always being broken down |
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What is a 2nd messenger?
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A low molecular weight, intracellular signaling molecules
ex. cAMP whose effect is mediated by regulation of PKA enzyme |
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What is protein kinase A (PKA)?
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- cAMP-dependent protein kinases
- it phosphorylates other target proteins at serine/threonine kinases - represent about 2% of the human genome |
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What components does an inactive PKA have?
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- tetramer with 2R and 2C
- regulatory subunit (R) which as a nucleotide binding site - catalytic subunit (C) bound to psuedo substrate site |
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How does PKA get activated?
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- cAMP binds to nucleotide binding sites, conformational change of R
- C lets go and catalytic sites become available |
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What is cooperative allostery?
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- binding of first cAMP reduces the Kd for binding of the second
- small changes in [cAMP] c= large changes in the [active PKA] |
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What does PKA do?
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- release glucose which can be a source of ATP for muscles
- turn off (phosphorylates) glycogen synthase and on (activates) phosphorylase |
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How is energy made in the muscle?
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- glycogen --> Glucose-6-Phosphate (G-6-P)
- G-6-P enters glycolytic pathway metabolized to ATP = energy for muscle contraction |
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How is energy made in the liver?
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- glycogen degradation to G-6-P is enchanced
- glycogen synthesis is inhibited (phosphorylation and inactivation of glycogen synthase) - glycogen degradation by activation of glycogen phosphorylase - G-6-P --> free glucose - glucose is released to blood & transported to other tissues |
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How do bacterial toxins affect cAMP levels?
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- chemically modify Gsa-GTP so that it is non-hydrolyzable
- it stays activated even w/o adrenaline stimulation - adenylyl cyclase stays active, cAMP levels rise |
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Why is increase in cAMP good for bacteria?
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relaxation of gut, loss of water and electrolytes, watery diarrhea
--> dispersal to other hosts |
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How are toxins and their effect on cAMP levels medically relevant?
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- since they target the GSA subunit, we can now DIRECTLY combat the bacteria with drugs
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How is gene transcription activated with GPCRs?
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- free catalytic subunits of PKA (C) phosphorylate and activate transcription factors like CREB
- binds to CRE (cAMP regulatory element) |
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What is CRE and what does it do?
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- regulatory element of various target genes
- genes for enzymes required for glucose production |
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GPCR are the most versatile class of receptors. T/F?
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True.
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Outline the general GPCR pathway.
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- receptor interacts with a ligand (signal) outside cell
- change in receptor conformation - signal transduction in cell via interaction w/ G-protein |
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What is an agonist?
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Chemical analog that binds to the receptor and mimics the natural signal and accomplishes the same thing as the original signal.
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What is an antagonist?
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A chemical that interferes with the natural signal by competing for receptor binding. It binds receptor but does not induce hormone response (acts as inhibitor)
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Define exogenous.
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Something not native to the system, something you’ve added
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What is an example of an agonist?
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Isoproterenol which mimics epinephrine’s relaxing effect on bronchial SM by opening air passages
--> treatment of bronchial asthma and chronic bronchitis |
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What is an example of an antagonist?
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Alprenolol which has a high affinity for epinephrine receptors on cardiac muscle cells
- inhibits receptor signaling = reduces contraction rate - beta-blockers and treatment of cardiac arrhythmias |
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What is allosteric modulation of GPCR?
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Changing one part of the receptor which affects conformation of distant domain of receptor and alters its activity
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What is the specific target cell of the HIV virus?
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T-lymphocytes
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What is gp120 and what does it interact with?
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- a capcid protein on the HIV virus
- interacts with CD4 (a single transmembrane protein) and CCR5 (7 transmembrane GPCR) |
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How does HIV virus attack the cell?
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- gp120 is recognized by receptors
- virus interacts with CD4 and CCR5 and fuses with membrane - genome can then enter the cell |
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What happens to people without a functional CCR5?
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They do not show symptoms of HIV virus and are resistant to it
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What is the goal to prevent HIV virus from activating?
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Find an allosteric modulator that holds CCR5 in a conformation that makes it resistant to binding with gp120
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Why isn't finding a drug that blocks the gp120-CCR5 interaction easy?
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Viruses adapt, mutations can happen, the allosteric modulator can be defeated
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Why is receptor internalization helpful?
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Without a receptor, the signal is blocked from delivery into the cell. This is useful for stopping viruses from entering the cell, ex. internalization of CCR5 receptor to stop HIV virus.
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How can you stop constitutively active receptors?
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Alter the G-protein interaction domain using an inverse agonist.
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What do inverse agonists do?
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They bind to secondary sites and can change receptor conformation so that it doesn't interact with G-protein anymore (silences the interaction).
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What's the difference between agonist and inverse agonist?
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Agonist - turns something normally off, on
Inverse agonist - turns something off that is normally on |
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Why are plants being used as G-protein model systems?
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They have few GPCRs compared to humans.
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What is an example of a plant species used in G-protein research?
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Arabidopsis.
Hormone/signal = abscisic acid (ABA) |
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What does ABA do?
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- mediates aspects of plant growth, development, response to environmental stress
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Which responses are mediated by ABA?
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- bud dormancy
- seed maturation and dormancy - promotes abscission (dropping of leaves in winter & fruit in summer) - inhibits seedling (growth depending on environment) - triggers closing of guard cells in response to drought |
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Where and when is ABA synthesized?
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In the roots of plants and when water levels are low in soil
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When guard cells need to be closed this suggests that...
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ABA will be produced and there must be low water in soil.
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What happens during stomatal closing?
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1. K+ ions move out of the vacuole and cells.
2. Water follows out. 3. Guard cells shrink in size. 4. The stoma closes. |
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What happens during stomatal opening?
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K+ ions move into the vacuoles, water moves into vacuoles, guard cells expand and stoma opens.
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What is the mutation that Arabidopsis Ga subunit (GPA1) have? How is this significant?
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They cannot close their guard cells.
--> G protein signaling is important in the ABA guard cell signaling pathway |
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What other GPCR signaling components were found in plants? What does this suggest?
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- G-protein cycle (guanine nucleotide exchange and GTP hydrolysis)
- few G protein effector proteins Cell division & ion channel regulation are processes regulated by G proteins in both plants and animals! |
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How was the first receptor for ABA recognized?
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They searched the Arabidopsis genome and found a gene (GCR2) encoding a putative GPCR. They looked at a hydropathic profile to see if any of the AA had a 7 transmembrane shape which GCR2 did.
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How was GCR2 confirmed as the ABA receptor?
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Mutated GCR2 gene --> mutant plants couldn't close guard cells even w/ ABA.
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How was ABA confirmed as the ligand for GCR2 receptor?
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GCR2 receptor binds ABA with high affinity at physiological levels typical for these cells. Also when ABA binds to the GCR2 receptor, GPA1 (apha subunit) disassociates.
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What does CAMs stand for? What do they do?
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Cell adhesion molecules allow cells to segregate into distinct tissues.
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Why is cell adhesion important?
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Cell-cell interactions are stabilized, communication is promoted between adjacent cells
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Name four types of CAMs.
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cadherins, Ig family, integrins and selectins
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How do epithelial cell sheets adhere to one another?
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They adhere at lateral sides, they have a basal and apical surface.
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Describe tight junctions.
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They are a network of proteins in a specific form that form junctions. Two cells are really closely attached to one another and junctions where the are pinned together are where the proteins are fused.
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Why and how are tight junctions beneficial?
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Nothing can be passed through adjacent cells through junctions. This is proved by adding lanthanum hydroxide (stain) at basal surface which did not reach apical surface. This is good so that blood does not just randomly flow out of vessels.
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Describe gap junctions.
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In gap junctions, a collection of proteins lie on the lateral surfaces and communication can occur through connexons.
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2nd messengers and ions cannot pass through gap junctions. T/F?
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False.
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Describe adheren junctions.
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They allow specificity; help like cells hold together. Cells adhere only to other cells expressing the same cadherin = homophilic interactions.
ex. E-cadherin joins two epithelial cells together |
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How are cadherin proteins linked to signaling pathways?
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They are linked to catenin proteins which are part of signaling pathways
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What observations can be made when you fuse GFP and E-cadherins?
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Increase in expression of GFP at the junctions where cadherin proteins are accumulating. If you add a neural cell then you would not see the GFP at the junctions because cadherins would not accumulate together.
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What is extravasation?
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Leukocytes move out of blood vessels into damaged or infected tissues.
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What are granulocytes?
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- has toxins for killing pathogens
- neutrophil, eosinophil, and basophil |
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What are monocytes?
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They are macrophages that undergo phagocytosis and extravasation.
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What are lymphocytes?
*= extravasation |
NK cells* - Lysis of virally infected cells and tumour cells
T and B cells* - immune response |
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What are the 5 steps of extravasation?
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Capture, rolling, slow rolling, firm adhesion, transmigration
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Where is the cell adhesion molecule p-selectin found?
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In the vacuole of epithelial cell (ex. in one from blood vessel wall)
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Where is ICAM-2 found?
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It is a cell adhesion molecule found outside, on the epithelial cell membrane.
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What is a selectin ligand and where is it found?
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It is found on a neutrophil or leukocyte, and it is a specific carbohydrate like glycoprotein.
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What components are on a WBC in the inactive state?
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1. selectin ligand
2. PAF receptor 3. aLB2 integrin |
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What is the PAF receptor responsible for?
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It helps with signaling.
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How does p-selectin or e-selectin come out of the vacuole?
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TNFa signals the vacuole to release these cell adhesion molecules on the surface of the cell.
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What happens during the rolling adhesion of extravasation?
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- p-selectin and e-selectin interacts with carbohydrate groups (selectin ligand) on WBC
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What happens during the slow rolling part of extravasation?
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- increase in p-selectin expression to slow down movement some more
- rolling occurs due to weak adhesion of selectin and leukocyte |
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What is tight binding or firm adhesion mediated by?
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Integrins on WBC
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What is IL-8 (PAF)? Where is it located?
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It is a membrane anchored protein that acts as a signal on the membrane of the epithelial cell. It is always there but only needed during firm adhesion.
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What does the IL-8 (PAF) signal do?
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It interacts with the cytokine PAF receptor on the leukocyte and signals it to activate integrins.
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How are the integrins on the leukocyte activated?
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They are activated when the PAF signal on the epithelial cell interacts with the PAF receptor on the leukocyte.
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What do the integrins do?
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Integrin receptors bind to ligands on the epithelial cell called ICAMs to allow for firm adhesion.
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What are ICAMs?
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Intercellular adhesion molecule.
They are ligands that bind to integrins on leukocytes during firm adhesion. |
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How does firm adhesion or tight binding during the extravasation process occur?
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Interaction of ICAM-1 and integrin on leukocyte surface.
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Why is the ICAM/integrin binding necessary?
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To stop the motion of the rolling leukocyte.
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What does PAF stand for?
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Platelet-activating factor
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What happens during transmigration?
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WBC changes its shape and interacts with the endothelial cells to get through the membrane.
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What is p53?
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A tumour suppressor gene
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What is B-cell lymphoma?
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Cancer of the blood and lymph nodes
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Where is B-cell lymphoma most prevalent?
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Central band of sub-Saharan Africa = "lymphoma belt"
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What are the two main events of cell cycle?
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1. Chromosome replication (DNA synthesis)
2. Cell division |
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What happens if you lose control of the cell cycle?
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It can lead to cancer
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What are the five phases of cell cycle?
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G0, G1, S, G2, M
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What happens in the G1 phase?
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Growth and metabolic events
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What cells can be in G0 phase? Which ones can't?
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Precursor or stem cells that proliferate, not muscle, skin or neural cells.
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What happens at the end of S phase?
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Sister chromatids are replicated
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Daughter cells don't have to be be identical in terms of organelles, but DNA content has to be exactly replicated, even in terms of DNA modifications. T/F?
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True.
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What are the five stages of mitosis?
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Prophase - chromsomes condense, nuclear envelope breaks down, spindle assembles
Prometaphase - chromosome attaches to spindles Metaphase - chromosomes align at center Anaphase - sister chromatids separate to opposite spindle poles Telophase - chromosomes decondense, nuclear envelope reforms, spindles disassemble |
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How is the cell cycle controlled?
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Regulated phosphorylation and degradation
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What are Cdk-cyclin complexes?
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They are heterodimeric kinases made up of a regulatory subunit - cyclin and catalytic subunit - cyclin-dependent kinase
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What is necessary for regulated phosphorylation in cell cycle?
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Cyclin-cdk complex
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What happens during regulated degradation?
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ubiquitination of cyclins and proteolysis
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What is needed for degradation to occur during cell cycle? Name 2 examples.
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E3 ligase complexes
ex. APC and SCF |
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What are the major classes of Cylin-cdk complexes?
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G1 cyclin-CDK, S-phase cyclin-CDK, mitotic cyclin-CDK
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What do G1 Cyclin-Cdk complexes do?
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- prepare cells for S phase
- induce synthesis of proteins required for DNA synthesis - induces degradation of S-phase inhibitors --> activating S-phase cyclin-Cdk complex (stimulate cell entry into S-phase) |
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What are the targets of phosphorylation for G1 cyclin-CDK?
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- S-phase inhibitors
- transcription factors required for synthesis of S-phase proteins |
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What are the degradation targets for G1 cyclin-CDK?
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- S-phase inhibitors
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Why are S-phase inhibitors needed?
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To ensure that cell division doesn't happen too soon
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What do S cyclin-Cdk complexes do?
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- activate and assembly of pre-replication complex so DNA replication can begin
- prevents more pre-replication complexes from being activated |
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Why is it important to prevent reassembly of pre-replication complexes? What makes sure of this?
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Prevents another round of DNA synthesis, so replicate each chromosome only once. S cyclin-CDK complexes do this.
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What are the phosphorylation targets for S cyclin-CDK?
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- proteins of pre-replication complex
- transcription factors required for synthesis of mitotic proteins |
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What does APC stand for? What is it?
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Anaphase promoting complex, it is an E3 ligase.
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What are the targets for phosphorylation of mitotic cyclin-CDK?
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- proteins required for:
chromosome condensation breakdown of nuclear envelope assembly of mitotic spindle apparatus alignment of chromosomes at metaphase plate - activates APC at different steps |
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What are the targets for degradation of mitotic cyclin-CDK?
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- cyclins
- anaphase inhibitors |
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What does MPF stand for?
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Maturation promoting factor
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What is MPF?
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Heterodimer of mitotic cyclin B and CDK
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What species were experimented to find cyclin-CDK complexes and what was found?
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Mammalian cells and Xenopus laevis oocytes.
- MPF was found |
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What species was experimented to find cyclin and what was found?
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Sea urchin embryos and cyclin B (a cyclically regulated protein) was found in high peaks right before mitotic divisions
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Describe the experiment conducted to find cyclins?
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Using fertilized sea urchin embryos that entered into mitosis continuously, they isolated proteins at various time points along process and ran Western blot and found cyclin B.
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How did they find out that kinase activity was linked with cyclin levels?
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Two assays in Xenopus laevis:
1. To detect kinase activity - they checked for phosphorylation of H1 histones (chromatin proteins) 2. Cyclin levels - antibody to Cyclin B |
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What happens when RNAase is added to the cell free extract?
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RNAase = removes mRNAs = no proteins
- no kinase, cyclin B or mitotic events present - shows that kinase IS needed for mitotic events |
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What happens when you have RNAase + cyclin B mRNA?
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Mitotic events can occur normally, this shows that when cyclin B is present, kinase activity follows.
So cyclin B regulates kinase activity! |
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What does it mean to be a non-degradable cyclin B protein?
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It doesn't have the deadbox component regonized in the ubiquitinalation process
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What happens when you have RNAase + non-degradable cyclin B in the cell free extract?
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Cells enter mitosis normally, but once they reach the peak, chromosomes don't decondense because cyclin B is not degraded and kinase stays high as well.
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Cyclin B needs to be degraded for a cell to exit mitosis. T/F?
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True
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Cyclin B levels are high at the end of mitosis. T/F?
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False. They are high at the end of G2 throughout M, but go down at the end of M.
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Why are yeast useful models for cell cycle mutation studies?
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- fewer proteins involved in regulatory pathways
- grow quickly, single celled, mutations can be created - temperature-sensitive studies can be performed |
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How do genetic screens work?
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- organisms are mutagenized using radiation and chemical mutagens at random
- screen looks like each phenotype of mutation for that in interest |
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What is a temperature sensitive mutant?
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It is a conditional mutant.
This mutation in a gene codes for a protein that folds and functions at normal (permissive) temp. but not at elevated (restrictive) temp. |
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How are temperature sensitive mutations different from WT ones?
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WT gene will code for a protein that folds at normal and elevated temperatures. TS ones only at permissive temperatures.
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Why are temperature sensitive (TS) mutations useful?
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- yeasts are haploids, in these organisms a mutation can easily be lethal so TS mutations help to study loss of function mutations
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What does recessive mean for TS mutants?
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absence of wild-type function
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What does dominant mean for TS mutants?
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increased wild-type function
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What does cdc stand for?
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cell-division cycle mutants
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What kind of yeasts are S. pombe and S. cerevisiae?
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fission and budding
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How do researchers perform a screen to isolate TS mutants?
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- mutagenize yeast w/ chemicals or radiation
- yeast will now be randomly mutated at diff. genes - mutated yeast are grown at 25 degrees - increase to 35 degrees - find those that can't divide or divide too quickly |
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How is yeast division different from regular cell division?
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- actin rings to divide
- nuclear envelope doesn’t break down but the spindle forms outside and comes into the nucleus to attach onto the chromosomes |
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What phenotypes did the cdc's for S. pombe have?
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- tiny cells (wee) --> divided too quickly, don't grow large enough when entering M phase
- elongated --> cells grew too much, cannot enter mitosis |
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Describe the recessive mutation for Cdc2.
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cdc2- loss of function mutation in Cdc2 protein function
It fails to undergo mitosis --> elongated cells |
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Describe the dominant mutation for Cdc2.
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cdcD - gain of function mutation in Cdc2 protein function
Premature mitosis - wee cells |
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What happens if we lose Cdc2?
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Cells fail to divide and enter into mitsos. Cdc2 is a positive regulator of mitosis.
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What is Cdc2?
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A gene that codes for a protein with kinase activity and a key regulator of entry into mitosis.
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What is Cdc2 a homolog of?
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CDC28 in S. cerevisiae
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What does Cdc2 form a heterodimer with?
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Cdc13
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Why is Cdc13 required?
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For entry into mitosis (as well as Cdc2)
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What is homologous to Cdc13?
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Cyclin B in Xenopus
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What is the MPF in S. pombe species? What else does it act as?
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Cdc2-Cdc13 heterodimer. It is also the M, G and S phase cyclin-CDK
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Does Cdc25 activate or inhibit MPF?
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Cdc25 stimulates mitosis by activating MPF.
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Does Wee1 activate or inhibit MPF?
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Wee1 inhibits mitosis by inhibiting MPF.
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What are the phenotypes of excess and deficient Cdc25?
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wee (deficient G2) and elongated cells (increased G2)
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What are the phenotypes of excess and deficient Wee1?
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Elongated (increased G2) and wee (decreased G2) cells
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How does Wee1 inhibit MPF?
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It has a tyrosine kinase, phosphorylates Tyr15
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How does Cdc25 activate MPF?
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It has a phosphotase that dephosphorylates Tyr 15.
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What are the steps of MPF regulation of S. pombe?
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1. Mitotyic cyclin and CDK combine to make inactive MPF
2. Wee1 phosphorylates Tyr15 to keep it inactive 3. CAK phosphorylates T161 and it stays inactive 4. Cdc25 dephosphorylates Tyr15 and the MPF becomes active |
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Why is CAK needed?
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CAK protein binds much more efficiently to our MPF that is phosphorylated at T than the one that is not.
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What is unique about budding yeast division?
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It begins cell division even before it begins mitotic division, the buds will form or daughter buds will come off the mother one. No envelope break down in these species, spindle poles are outside envelope.
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What phenotype are we looking for in S. cerevisiae?
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arrest of budding at non-permissive temperature
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If you add CDC28 to a pombe cell that has a lack of function mutation for Cdc2 what happens?
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Cells go from elongated to normal because Cdc2 and CDC28 are homologous.
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What's the difference between Cdc2 and CDC28?
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cdc28 mutants are arrested in G1 while cdc2- in G2.
Cdc2 for entry into Mitosis & Cdc28 needed for entry into S phase |
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When do arrests happen in CDC28 mutants?
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In G1, before S phase, because in cerevisiae cells, the cell buds before replication.
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Describe how to identify the protein CDC28 using functional complementation.
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Start off with a cdc28 TS mutant gene that cannot divide or grow at restrictive temperatures. Using actively dividing cells, you create a cDNA library w/ only cell cycle regulatory genes.
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What does it mean if you add a gene that causes the Cdc28 TS mutant to divide under non-permissive temperatures?
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That gene Z must be the WT copy of the cdc28 gene.
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What happened after they isolated the circular vector of gene Z?
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They sequenced and called it CDC28. They realized it was similar to it's homolog cdc2.
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What can functional complementarity techniques be used for?
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To find homologs of certain genes or finding those responsible for cell cycling.
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What is SPF and what is it made of?
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S-phase promoting factor, made up of CDC28 (Cdkinase) and cyclin.
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What does SPF do?
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phosphorylate & regulate proteins required for DNA synthesis
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How many cyclins does pombe vs. cerevisiae have?
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1 vs. at least 6
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What are CLN1,2,3 and CLB1,2,3?
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Both are types of cyclins in S. cerevisiae. CLN makes up the SPF and combines with the CDC28 kinase and helps transition from S-phase to G, CLB helps entry into mitosis.
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What is required for unidirection progress through mitosis AND inactivation of cyclin-Cdk complexes?
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Protein degradation
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What does APC stand for?
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Anaphase promoting complex
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What is APC?
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A multi-protein complex w/ a ubiquitin ligase subunit
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When and how does APC get activated?
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When MPF levels are elevated and by phosphorylation
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What are the two substrate-specificity factors of APC?
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Cdc20 and Cdh1
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What needs to be degraded after the onset of anaphase? What happens to them?
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Anaphase inhibitors (by APC-Cdc20) and mitotic B-type cyclins (by APC-Cdh1) are ubiquitinylated and targeted to the proteosome
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What is common about all B-cyclins which is where are they degraded?
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All contain a homologous sequence near the N-terminus called destruction-box
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What does the sequence for D-box look like?
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RxxLxxxxN/Q
where x is any amino acid, R is Arginine L is Leucine and N/Q is Asparagine or Glutamine. |
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What happens if the d-box is not present on the cyclin-B protein?
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The cyclin cannot be degraded
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What happens if the d-box is ADDED to a protein?
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It will be degraded during the transition out of mitosis
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What stages of the cell cycle does APC regulate?
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Anaphase entry and mitosis exit
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What would happen if the anaphase inhibitors were not degraded?
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The cell would stay in metaphase and never transition into anaphase
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When is APC-Cdc20 active?
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After metaphase, when the cell is entering anaphase
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When is Apc-Cdh1 active?
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When the cell needs to exit mitosis, so after telophase
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What are the levels of mitotic cyclin and MPF like at metaphase?
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Both high
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What are the levels of mitotic cyclin and MPF like at telophase?
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Both low
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What is SCF and what does it target?
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It is another ubiquitin ligase complex, it targets Sic1 for degradation.
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When is Sic1 targeted for degradation and by what complex?
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Between G1 and S phase transition, by SCF ligase
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What is Sic1?
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It is an S phase inhibitor
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What is the process of degradation for entry into S phase?
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In mid-late G1, Sic1 is phosphorylated by G1-cyclin-CDK. It is polyubquitinylated by SCF and degradation by proteosome. Cell goes into s-phase and DNA replication occurs.
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Post-mitotic cells are those that divide and undergo mitosis. T/F?
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False. They are fully differentiated cells and never re-enter the cycle, stay in G0 phase.
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Cell Cycle mechanisms in yeast and Xenopus also operate in mammals. T/F?
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True.
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How do mammalian cells differ from yeast cells in terms of Cdks?
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Mammalian cells have multiple (1,2,4,6), yeast only one.
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What are Cdk1 and Cdk2 in mammalian cells homologs of?
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Cdk1 --> S. pombe Cdc2 (acting as MPF)
Cdk2 --> S. cerevisiae Cdc28 (acting as SPF) |
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Mammalian cyclins and cdk proteins are expressed in G0. T/F?
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False.
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What are mitogens and what do they do?
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- extracellular growth factors that induce synthesis and activity of G1 cyclin-Cdk complexes
- initiate entry to cell cycle |
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Describe the relationship between mitogens and restriction points.
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The growth factor mitogen activates the cell and sends it into G1. Taking away mitogen causes the cell to recede back into G0. Once it passes the RP, taking away the mitogen will do nothing.
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What is a restriction point?
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Point in late G1 where passage through cell cycle becomes independent of mitogen presence.
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Define quiescent cells.
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Cells in G0 phase.
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What happens when you add growth factors to G0 cells?
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Transcription occurs
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What are early-response genes important for?
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They encode transcription factors needed for transcription of delayed-response genes
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What happens when serum + protein-synthesis inhibitors are added to G0 cells?
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No ER proteins are produced, so no ER transcription factors, no transcription of DR genes. Also there are no ER proteins to down regulate ER genes. Cells essentially cannot divide or enter cell cycle.
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Why are early response genes still activated when protein synthesis inhibitors are added?
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They get activated by transcription factors (like TCF and SRF) that are already present and get phosphorylated by MAPkinase. This leads to transcription of genes like c-fos (ER gene).
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If c-fos is an early response gene that suggests that...
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c-fos gene will code for TF needed for transcription of delayed response genes (DR genes)
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Describe the mammalian cell cycle in terms of when cyclins are active.
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- cyclin D during G1
- cyclin E for entry into S phas - cyclin A during S phase to make sure DNA replication is completed - cyclin A and B in G2 and are at high levels in M phase |
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What do DR (delayed response) genes code for?
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E2F transcription factors
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What is the restriction point regulated by?
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E2F transcription factors and Rb protein
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What do E2F transcription factors do?
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- activate transcription of cyclin E, G1 to S phase cyclin and stimulates DNA synthesis
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What does Rb protein stand for?
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Tumor-Suppressor Retinoblastoma
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When Rb is bound to E2F, then E2F is active and cell passes through G1 phase. T/F?
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False. Rb + E2F = inactive, cell cannot go from G1 to S phase.
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How is Rb inactivated?
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Phosphorylated by G phase cyclin-Cdks (Cyclin D - Cdk 4/6)
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At the restriction point, what is there a high level of?
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Cyclin E - Cdk2 complex to reach the RP.
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Progression through and exit out of mitosis requires a decrease in...
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mitotic cyclins and MPF
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When MPF is removed, what component takes over and why?
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Phosphotases - reverse all the phosphorylations that helped the cell get into mitosis
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What happens to MPF and target proteins at the end of mitosis?
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MPF is inactivated through cyclin degradation and all same target proteins are dephosphorylated
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Which proteins are required for chromosome condensation at prophase?
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condensins and topoisomeraser II
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What does condensin do and what is it made up of?
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It is made of up 5 proteins called Smc proteins. It loops strands in one DNA molecule together, condensing them along the longitudinal axis.
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What do cohesion proteins do?
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loop two replicated DNA molecules
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What do cohesion and condensin proteins do and when?
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They help in the supercoiling of DNA during prophase
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When are condensins and Topo II activated and how?
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Beginning of prophase - phosphorylation by MPF (CyclinB/CDK)
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Condensins are present during interphase. T/F?
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False. Only cohesion is, which gets replaced by condensin during propase
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What is the purpose of Topo II?
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To ensure that DNA loops do not get looped and tied together by accident.
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When does chromosome decondensation occur?
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After metaphase and anaphase, as they enter into telophase.
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What components does chromosome decondensation require?
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- activity of phosphotases
- absence of kinases (inactivation of MPF or CyclinB/CDK) |
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What happens if you add G1 extracts to mitotic chromosomes?
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They begin to decondense.
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In the absence of cyclin B degradation, chromosomes cannot decondense and mitotic spindles can’t degrade. T/F?
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True.
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What does APC-Cdh1 target for degradation?
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Cyclin B or mitotic cyclin
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What happens in the cell when APC-Cdh1 targets cyclin B?
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Low MPF activity thus chromosome decondensation and disassembly of the spindle
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When and how do cohesin proteins assemble onto unreplicated chromosomes?
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During telophase and G1, the complexes form rings
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How do cohesion proteins assemble on replicated DNA molecules?
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They form rings by the end of G2 on unreplicated molecules, then replication occurs in the rings and stays in this form from G2 to metaphase.
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How and when are the cohesin rings destroyed?
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At anaphase, by cutting one ring will break all the rings.
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How are cohesin molecules released in two stages?
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Prophase: cohesion molecules lie along the length of the chromosomes
Metaphase: protein is phosphorylated and released by kinases Anaphase: remaining cohesin at the center is cleaved to release sister chromatids |
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What is securin?
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An anaphase inhibitor
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What happens when securin is deactivated and when does this happen?
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During the transition of metaphase and anaphase, degradation of securin leads to activation of separase and proteolysis of cohesin
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How does APC-Cdc20 cut the cohesin complex?
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1. Separase and securin (anaphase inhibitor) are combined
2. APC-cdc20 targets securin and breaks it down 3. Separase cleaves cohesin complex |
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When and how do cyclin B and securin get deactivated?
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cyclin B - telophase, end of mitosis by Cdh1
securin - metaphase to anaphase transition by Cdc20 |
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What is made up of the nuclear envelope?
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It is double layered, so nuclear lamina on inside
Nuclear lamins (intermediate filaments) polymerize into filaments Lamins A, B, C |
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How does the nuclear lamina disintegrate?
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Lamin filaments are phosphorylated at serine residues --> depolymerization of all lamins except lamin B which stays on nuclear membrane
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Which lamin does not depolymerize along with others and stays on nuclear membrane instead? Why?
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Lamin B - it has an isopropyl group, depolymerization requires phosphorylation at serine residues
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What experiment helps see dependency of phosphorylation of lamins in nuclear lamina disintegration?
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Replace serine groups on lamin A with alanine groups so it can no longer be phosphorylated
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What is Cdc14 and what does it do?
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It is a phosphotase and reverses the effect of MPF.
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When does Cdc14 dephosphorylate lamin proteins?
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When there's no MPF or mitotic cyclin, late anaphase.
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What happens in the cell when Cdc14 acts?
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- nuclear membrane proteins are dephosphorylated
- ER membrane and nuclear membrane proteins associates with the chromosomes |
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Define karyomere and when they are formed
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When chromosomes form it's own mini envelope when Cdc14 reverses the effect of MPF
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Where can nuclear envelope assembly be seen?
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Nuclear envelope assembly in Xenopus extracts (in vitro).
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What mediates the phosphorylation of proteins during mitotic events?
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MPF
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Why is protein degradation necessary and which are the main proteins being degraded?
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For unidirectionality of events.
cohesin - metaphase to anaphase cyclin B - end of anaphase, exit from mitosis |
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What is the active cyclin/cdc2 responsible for?
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phosphorylation of:
condensins - condensation lamins - NEB membrane proteins - dissociation |
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Why is deactivation of cdc2 necessary after mitosis?
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allows dephosphorylation and reversal of mitotic events
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Why are cell cycle checkpoints needed?
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Ensure that the genome is intact and that each cell cycle phase is completed before proceeding to next phase
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What happens if there is an error during cell cycle checkpoints?
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Delay progression until error is corrected
Induce apoptosis if problem cannot be fixed |
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What can cause S phase arrest?
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unreplicated DNA
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What can cause G1 or G2 phase arrest?
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DNA damage
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What can cause M phase arrest?
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Improper spindle formation
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What is the pathway for detecting DNA damage at G1 arrest?
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ATM - recognizes cut double stranded DNA typical of IR radiation, shorter than normal
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What is the pathway for detecting DNA damage at G2 arrest?
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ATR - recognizes a dimer called thiodine and binds to the damaged, dimerized DNA
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Outline the ATR pathway.
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1. ATR which is a kinase (activated when bound to DNA) phosphorylates Chk1 (another kinase) and activates it.
2. Chk1 phosphorylates Cdc25 (a phosphotase) and is held by adaptor proteins so that it cannot dephosphorylate Cdk and turn on MPF 3. Cell arrests in G2 |
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What protein holds Cdc25 that has been phosphorylated outside in cytoplasm?
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An adaptor protein
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Outline the ATM pathway.
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1. ATM binds to kinase Chk2, which phosphorylates p53 (a transcription factor) that gets stabilized.
2. p53 binds upstream on p21 gene, which gets activated and transcription occurs. 3. p21 protein is formed (S phase inhibitor) and binds to Cdk (s phase promoting complex). 4. Cell is arrested in G1 and waits for damages to be repaired |
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Why is p53 transcription factor very unstable?
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It is polyubiquitinylated by ubiquitin ligase and degraded by proteosome.
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What helps stabilize p53?
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Phosphorylation by Chk2
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How is p53 useful if cell damage is so severe?
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It induces apoptosis by activating the expression of pro-apoptosis genes
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Which genes does p53 activate the expression of?
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Cdk inhibitor - p21(CIP) and pro-apoptosis genes
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Name two types of Cdk inhibitors that p53 activates the expression of.
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p21 - CIP and p27
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What is the protein that forms when the gene p21 is translated?
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CIP protein
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What does p21 do?
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It binds to cyclin-Cdk complex, inhibits kinase activity and arrests it in G1 or G2.
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What does the Cdk inhibitor p27 interact with?
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It interacts with Cdk2 (complex with cyclin A) and blocks substrate binding site for that complex so it stops the kinase activity
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What happens at the unreplicated DNA checkpoint?
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ATR associates with the replication fork, activating the kinase Chk1 which inhibits Cdc25 from activating MPF
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Why is it important that Cdc25 is inhibited by Chk1?
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So DNA replication can occur through to completion before the cell starts to divide
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Initiation of mitosis is independent upon completion of replication. T/F?
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False. Cdc25 is inhibited until the DNA is fully replicated.
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What needs to happen in the cell during the metaphase to anaphase checkpoint?
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All the chromosomes have to be attached to mitotic spindles before it goes through anaphase
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How were mutations in the spindle assembly checkpoint identified?
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Genetic screen for mutations that failed to arrest during cell division even if chromosomes had not attached
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What mutants were used in an experiment to test spindle assembly checkpoints?
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Yeast cells treated with benomyl (destabalizes microtubules)
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What happens when benomyl destablizes the microtubules?
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Microtubules are disrupted, spindles fall apart, chromosomes cannot attach, metaphase doesn't occur
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What is the name of an important spindle assembly checkpoint protein? What does it do?
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Mad2 - inhibits APC-Cdc20 (which initiates anaphase) and helps the cell stay in metaphase
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Can Mad1 protein attach to chromosomes if they are attached to spindles?
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No. This is called lack of attachment.
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What conformation are the Mad2 proteins in the cytoplasm?
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Open
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What is the role of Mad2 closed conformation?
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It binds directly to Mad1 and then promotes the C conformation in other Mad2 (O) proteins that attach to it
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What do Mad2 closed conformation proteins do?
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They bind to Cdc20 proteins to stop them from binding with APC which would initiate anaphase, but this way the cell stays in metaphase.
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It is sufficient for the checkpoint to be activated and cell to stay in metaphase if there are Mad2 proteins on ONLY one chromosome, as opposed to all 46 chromosomes. T/F?
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True.
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How does the spindle assembly checkpoint get inactivated? What stage does the cell go to then?
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Chromosomes attach to mitotic spindles and release the Mad1 and Mad2-C proteins. p31 can bind to free Mad2C proteins and convert them to open conformation which releases the Cdc20. Eventually last chromosome is attached to spindle and all Cdc20 is freed up, inactivation is complete.
Cell then enters anaphase. |
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What happens to Cdc20 when it is released after p31 binds to Mad2C?
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It can then bind to APC and form a complex to cleave cohesin molecules so cell can enter anaphase.
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What does Cdh14 do?
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It's a phosphotase, and dephosphorylates Cdh1 so it can bind to APC and become active and degrade mitotic cyclins.
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When is Cdh14 produced and where does it remain when it's not used?
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It's made during interphase and early mitosis, stays in nucleolus.
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When is Cdh14 needed?
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When the cell needs to exit from mitosis
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How is the chromosome segregation checkpoint maintained?
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via set of proteins called mitotic exit network (MEN)
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