BMB508 - Cell cycle II - G2/M transition and apoptosis

What cyclin initiates mitosis?

The mitotic cyclin-CDKs

- The mitotic cyclin-CDK gradually accumulate during S-phase

- The mitotic cyclin-CDKs are subdivided into cyclin A and B.

- During the assembly, mitotic CDKs complexes are maintained in an active state through inhibitory phosphorylation on CDK1 subunit.

What residues in the mammalian CDKs are subject to regulated phosphorylation?

Tyrosine and threonine residues

What controls the phosphorylation/dephosphorylating of CDK1?

Wee1 and Cdc25B and Cdc25C

The phosphorylation state of T14 and Y15 is controlled by a dual-specificity protein kinase known as Wee1 and a pair of dual-specificity phosphatases, Cdc25B and Cdc25C

When is the mitotic CDKs activated?

Until cells enter mitosis

It is inactive until it has reached the mitosis but will gradually accumulate during S-phase and G2 phase.

What is Wee1 kinase?

Dual specificity kinase that inactivates M-Cdk; primarily involved in suppressing Cdk-1 activity

What is Cdc25 phosphatase?

It activates the CDK complexes!

Cdc25A

•G1/S phase CDKs

•S phase CDKs

Cdc25B/C

•Mitotic CDKs

What does Wee1 and Cdc25 control together?

The entry of G2 cells into mitosis

What is the difference between Cdc25B and Cdc25C?

Cdc25B is thought to act as a starter phosphatase that initiates CDK1 activation.

While Cdc25C is the workhorse phosphatase that activates the vast majority of cyclin-CDK1 complexes.

What degrades the Wee1 kinase?

SCF! by doing the ubiquitin mediated degradation!

Marks wee1 for degradation so Cyclin B and CDK1 (mitotic cdks) wont be phosphorylated and deactivated again.

What does mitotic CDKs promote?

- Nuclear envelope breakdown during mitosis

- Condensation of the chromosomes during mitosis

What is PLK1?

Critical for the formation of the mitotic spindle as well as for chromosome segregation!

What is an aurora kinase?

They play a key role in the mitotic spindle formation and in ensuring that chromosomes attach correctly to the mitotic spindle so that they are segregated accurately during mitosis.

The aurora kinase family members are Aurora A and Aurora B

Why must the breakdown of the nuclear envelope happen during mitosis?

This is done to make the nuclear chromosomes interact with the cytoplasmic microtubules nucleated by the centrosomes

Chromosomes are surround by the nuclear envelope (in the nucleus)

Centrosomes that nucleate the mitotic spindle are located in the cytoplasm.

How does this nuclear envelope breakdown happen?

It is caused by the mitotic CDKs since they phosphorylate the serine residues in all three nuclear lamins A, B and C.

- This phosphorylation, together with phosphorylation of lamin B by protein kinase C, causes depolymerization of the lamin intermediate filaments

- Depolymerization of the nuclear lamins leads to disintegration of the nuclear lamina and contributes to disassembly of the nuclear envelope

M-CDKs => Phosphorylating lamins => Deploymerization => disintegration of the nuclear lamina.

What else can the mitotic CDKs also affect on the nuclear envelope?

Nucleoporins

Causes the nuclear pore complexes to dissociate during prophase.

- Thought to decrease their affinity for chromatin and further contribute to the disassembly of the nuclear envelope.

What is the mitotic spindle and what is its function?

Also known as mitotic apparatus! The mitotic spindle is a structure that forms during cell division and separates duplicated chromosomes

The function of the mitotic spindle is to segregate chromosomes so that the sister chromatids separate from each other and are moved to opposite poles of the cell.

What is the mitotic spindle made out of?

Microtubules

- That attach to chromosomes via specialized protein structures known as kinetochores, which form at the central centromeric region of chromosomes.

What happens with the centrosomes? Are they coordinated with the cell cycle?

The centrosome cycle is coordinated with the cell cycle to form a bipolar spindle that can correctly segregate chromosomes in mitosis

What is an amphitelic attachment?

This type of attachment, in which the kinetochores of each sister chromatid are attached to microtubules coming from opposing centrosomes

What is the sensing mechanisms used by cells to detect incorrect chromosome attachment?

It is based on tension

When sister chromatids are correctly attached to microtubules, their kinetochores are under tension

Merotelic, syntelic, or monotelic attachment leads to insufficient tension at kinetochores, allowing the cell to distinguish these faulty forms of attachment from the correct amphitelic one.

How does the cell measure whether or not kinetochores are under tension?

It uses the aurora protein kinase (aurora A and aurora B) and their associated regulatory factors sense kinetochores that are not under tension and sever these microtubule attachments, giving cells a second chance to get the attachment right.

What is cohesin and what does it do?

It is a protein complex that holds the sister chromatids together at the centromere.

- Composed of two large coiled-coil SMC

What is a condensin and what does it do?

Central to the process of chromosome condensation

- Most eukaryotes have two closely related condensin complexes, called condensin I and II where

- Condensin II => Prophase

- Condensin I => Prometaphase

Like cohesins, condensins are composed of two large coiled-coil SMC protein subunits that associate through their ATPase domains with non-SMC subunits.

- When condensin function is lost in cells, chromosomes do not condense and sister chromatid tangles are not resolved.

What triggers anaphase onset?

Cleavage of cohesin at centromeres by separase triggers anaphase onset

- In the middle of prophase are the cohesins only in the middle.

Look at figure 19-27

What is separase?

A. A protein that marks a protein called securin for destruction.

B. A protein that is part of the cohesin complex.

C. A protein that destroys cohesin through its protease activity.

D. A protein that targets the mitotic cyclin for degradation.

C. A protein that destroys cohesin through its protease activity.

A protease known as separase cleaves the cohesin subunit Scc1 (Figure 19-28), breaking the protein circles linking sister chromatids.

What is securin?

Securin is a protein that keeps separase inactive. When securin is bound to separase, separase cannot attack the cohesin between sister chromatids.

Basically, the action of securin is to keep sister chromatids together.

How and when is the securin protein degraded?

Once all kinetochores have attached to spindle microtubules in the correct bi-oriented manner, the APC/C ubiquitin-protein ligase, directed by specificity factor Cdc20 ubiquitinylates securin

Is degraded by proteasomes (marked for degradation from APC/C)

What checkpoint is the phosphorylated APC/C (Cdc20) inhibited by?

The spindle assembly checkpoint pathway (M-checkpoint)

Ensures anaphase is not initiated until all of the chromosomes have achieved proper attachment to the mitotic apparatus

- Cdc20 is inhibited until every kinetochore has attached to microtubules and proper tension has been applied to the kinetochores of all sister chromatids

What is important for anaphase chromosome movement?

Decline in mitotic CDK activity

If mitotic CDK inactivation is inhibited, anaphase does occur, but it is abnormal

Anaphase spindle elongation and the events associated with exit from mitosis such as mitotic spindle disassembly, chromosome decondensation, and nuclear envelope re-formation require something to accomplish it? What is it?

Require the de-phosphorylation of CDK substrates.

In other words, the phosphorylation events that triggered all of the different mitotic events need to be undone for the cell to revert to the G1 state

What does cytokinesis do?

A process where the cytoplasm and organelles are distributed between the two future daughter cells.

What regulates the cytokinesis?

The small GTPase RhoA => Leads to cleavage furrow formation and division of the cytoplasm to form two completely separate cells when it is activated!

Function: The formation of the contractile ring assembly (this ring ensures that each daughter cell receives half of the genetic material) requires the small GTPase RhoA.

When active GTP-bound RhoA both stimulates formin-mediated F-actin assembly and promotes the action of myosin II

- RhoA itself is activated by a conserved Rho guanine nucleotide-exchange factor (GEF) called Ect2, which is itself tightly regulated by a multiprotein complex called Centralspindlin

What is DNA damage?

Single-strand break

Double-strand break

Bulky adducts

Base mismatches, insertions and deletions

Base alkylation

Explain the role of ATM in DNA-damage-activated responses that leads to cell cycle arrest?

DNA damage => ATM recruitment => activation of downstream effector proteins Chk1/2, MK2 => inhibition of Cdc25B/C by phosphorylation => block of activation of M CDK's => inhibition of entry to mitosis

DNA damage => ATM recruitment => activation of p53 => production of CKI p21 => inhibition of either G1 CDK's, G1/S phase CDK's, S phase CDK's or M CDK's

DNA damage => ATM recruitment => activation of Chk1/2, MK2 => inhibition of Cdc25A by phosphorylation => CDC25A becomes target for SCF degradation => inhibition of G1/S phase and S phase CDK's => inhibition of transition to S phase

Explain the role of p53 in DNA-damage-activated responses that leads to cell cycle arrest?

The p53 gene like the Rb gene, is a tumor suppressor gene, i.e., its activity stops the formation of tumors.

Stimulates production of CKI p21

Regulated by MDM2

Explain the role of MDM2 in DNA-damage-activated responses that leads to cell cycle arrest?

MDM2 binds to p53 to inhibit its transcription factor activities and catalyze its ubiquitination => target it for proteasomal degradation.

DNA damage => ATM recruitment => phosphorylation of p53 on a serine residue i N-terminus => p53 freed from MDM2 => stable p53 => activation of transcription of the gene for p21 => inhibition of CDK1/2/4/6 complexes => apoptosis, DNA repair and/or kept in cell arrest.

DNA damage => ATM recruitment à phosphorylation of MDM2 => inactivation => increased stabilization of p53.

In cytoplasm the level of MDM2 is regulated by ATR and p14ARH which isolates MDM2 in nucleolus, where it can't reach p53

Explain the role of p21 in DNA-damage-activated responses that leads to cell cycle arrest?

Cyclin kinase inhibitor (CKI) in the Cip/Kip family.

They bind to cyclin bound CDK's to inhibit their activity

If not removed from targets => no DNA replication or mitosis

p21 is important for for establishing G1 and G2 arrest as response to DNA damage

Explain the role of cdc25A/B/C in DNA-damage-activated responses that leads to cell cycle arrest?

They are phosphatases that can activate CDK complexes!

Cdc25A

- G1/S phase CDKs

- S phase CDKs

Cdc25B/C

- Mitotic CDKs

Why is the level of p53 critical for cell survival or cell death?

p53 normal function is to limit cell proliferation in the face of DNA damage.

- Low level of p53 = Cell survival! (proliferation factors, survival factors and growth factors => Low/constitutive stress)

- High level of p53 => Apoptosis! (DNA damage checkpoint activation, spindle assembly checkpoint activation => Prevention/repair of damaged cells)

How does stress increase the level of p53?

(1) DNA damage

(2) ATM/ART kinase activation

(3) Chk1/Chk2 kinase activation

(4) Phosphorylation of p53 => Activates p53

(5) Active p53 binds to regulatory region of p21 gene => activates the transcription and translation of it!

(6) p21 (CDK inhibitor protein) Inhibits G1/S-CDKs and S-CDKS complexes by binding directly to them!

What does ATR, ATM and DNA-PK?

ATR and ATM are kinases that can detect DNA damage.

DNA-PK can also repair DNA damage and detect it!

What happens with Cdc25 when there is DNA damage?

(1) DNA damage

(2) ATM/ART kinase activation

(3) Chk1/Chk2 kinase activation

(4) Chk1/Chk2 phosphorylates the Cdc25

(5) Cdc25 is now inactivated because of the phosphorylation => Can no longer phosphorylate the CDKs.

What is the link between DNA damage and apoptosis?

When there is DNA damage, a lot of proteins such as p53 gets activated, which are necessary to start apotosis!

Example!

- DNA damage and the activated p53 leads to the activation of Bid, Bim and others.

- These can inhibit the Bcl-2, so it will no longer bind to Bax/Bak

- Bax/Bak can now make a pore where they will pump cytochrome C out of the mitochrondria into the cytoplasm.

- Cyt C will bind to Apaf-1 which will activate the caspsase-9 initiator, which will then activate the effector protein caspase 3.

- This will lead to apoptosis

How are DNA damage repaired, when it is a double stranded break (DSB)?

Non-homologous end joining (NHEJ) or homologous directed repair (HDR)

What is nonhomologous end-joining repair?

It is a pathway that repairs double-strand breaks in DNA. It is called "non-homologous" because the break ends are directly ligated without the need for a homologous template

What is homology-directed repair?

Mechanism to repair double stranded DNA lesions, most common is homologous recombination, can only be used when there is a homologue piece of DNA present in nucleus

Homology directed repair (HDR) requires a homologous sequence to guide repair.

What happens when a cell death happens by apoptosis?

Chromatin compaction, condensation of cytoplasm which leads to:

- Breakup of nuclear envelope

- Nuclear fragmentation

- Blebbing

- Cell fragmentation

It is then phagocytosis

What is apoptosis?

Programmed cell death

Apoptosis: genetic regulated process which happens under development of illness => degradation of most cell components and NO leakage of intracellular components but are phagocytosed

Apoptosis does NOT attract cells of the immune system!

What is necrosis?

cell death

Necrosis happens when cells are damaged or exposed to extreme stressful situations (heat or no oxygen) or are infected by pathogens.

Explain why an immune response is typically induced by necrosis (or necroptosis) but not by apoptosis

Necrosis: cell swell up => pores in plasma membrane => intracellular contents are leaked => white blood cells are attracted (macrophages and other phagocytes) => inflammation response (swelling, heat and pain) => clear out necrotic cells and initiation of repair of tissue

Necrosis attract cells of the immune system while apoptosis DOES NOT!

What is a caspase protein?

A protease that degrades proteins!

What are trophic factors?

They are survival signals

They are required for cells in multicellular organisms to stay alive!

If they are not there, then the cell will activate a suicide program => Apoptosis!

What is necroptosis?

Programmed necrosis

Necroptosis is often triggered by extracellular cytokines such as tumor necrosis factor alpha (TNFα). Activation of this cell-death pathway frequently causes inflammation and contributes to the development of many human diseases, including nerve degeneration and atherosclerosis.

What is Apaf1 apoptosome?

Apoptotic protease activating factor-1 (Apaf-1) is a key molecule in the intrinsic or mitochondrial pathway of apoptosis, which oligomerizes in response to cytochrome c release and forms a large complex known as apoptosome

What is the function of caspases in apoptosis and the role of the activation cleavage site

Function of caspases:

Synthesized as inactive precursors

Enzyme that degrades protein by cleaving aspartic residues.

The initial death signal promote assembly of large platforms that bring multiple initiator caspases together => pairs of caspases form dimers => activate each other by proteolytic cleavage in their activation cleavage sites.

One initiator caspase activates many executioner caspases resulting in a cascade of caspase activity amplifying the initial death signal => Mediates apoptosis by proteolytic cleavage of target substrates containing the correct cleavage sites.

- Targets are often proteins which the nuclear lamina and cytoskeleton constitutes of. Cleavage => apoptosis

What is the caspase recruitment domain (CARD)

Facilitates assembly of the apoptosome in the intrinsic pathway.

-The Apaf-1 apoptosome contains 7 CARD's, 1 in each Apaf-1 monomer.

- In the heptamer 2 of these domains will bind to domains in 2 procaspase-9 molecules => stimulation of dimerization and activation of initiator protease

- 7 Cytochrome C can bind to it!

What is the death-effector domain (DED)

Facilitates assembly of the DISC complex in the extrinsic pathway

What is the plasma membrane biology in resting and apoptotic cells

'Eat me signal'?

The "Eat me signal" is when phosphatidylserine have been translocated from the inner leaflet to the outer leaflet caused by apoptosis. These lipids will now be able to bind to a set of receptors on the surface of macrophages and other immune system cells => Triggering phagocytosis of the cell or apoptotic body

- Caspase-3 = Cleaves both ATP11A and ATP11 => Inactivating them. This stops the phosphatidylserine from being transported from the outer leaflet to the inner.

. - Additionally, caspase-3 cleaves XKR8, and in so doing activates its function: the truncated XKR8 binds to two other proteins, dimerizes, and becomes an active translocase, shuttling all phospholipids, including phosphatidylserine, from one leaflet of the plasma membrane to the other.

What are extrinsic apoptosis pathway?

Apoptotic program triggered by the binding of an extracellular signal protein.

- When we get a "death signal" that binds to the death receptors!

What are an intrinsic apoptosis pathway?

Apoptotic program that depends on the release into the cytosol of proteins from the mitochondrial intermembrane space.

What does a survival factor/trophic factors?

It is an extracellular signal molecule that inhibits apoptosis.

Explain how the intrinsic pathway of apoptosis is activated

The intrinsic pathway is activated when:

- Bad is active => No trophic factors

- Puma is activated => DNA-damage

- Bim is active => Disruption of integrin signaling

What does the bad protein?

Bad

Presence of trophic factors

- activation of receptor tyrosine kinases

- activation of PI-3 kinase PKB pathway

- PKB phosphorylates Bad (a BH3-only protein)

- Phosphorylated Bad forms a complex with a cytosolic 14-3-3 protein

- inactivation of Bad

Inactivated Bad is unable to promote apoptosis, thus trophic factors normally inhibit apoptosis through this mechanism.

What does the protein Puma?

Puma

DNA damage triggers the activation of Puma (BH3-only protein)

Can either:

- Activated Puma => Bind to Bcl2

- Activated Puma => Bind directly to Bak or Bax

What does the protein Bim do?

Disruption of integrin signaling, such as when a cell is detached from its extracellular matrix, triggers the release of Bim

Released Bim => Bind to Bcl2

What happens next when either Bad, Puma or Bim have been activated in the intrinsic pathway?

Bax and Bak are pro-apoptotic proteins necessary induction of the insintric pathway.

Bak is placed in the mitochondrial membrane closely bound to the anti-apoptotic protein Bcl2. Once released à oligomer formation

Bax is primarily cytosolic but when bound to pro-apoptotic proteins it binds to the outer mitochondrial membrane à oligomer formation.

Oligomer pores

- release Cytochrome c form mitochondrion to cytoplasm

- binding to Apaf-1

- activation of caspase-9

- cleavage of effector caspases

- activation of these

- degradation of cell proteins and cell death

How is the extrinsic pathways of apoptosis activated?

Activated by Fas, TNF-alfa or other proteins from the TNF-alfa family.

Death signal from a cell bind to death receptor on a nearby cell

=> binding of cytosolic death domains containing TRADD and RIPK1 to the death domain (DD) on the intracellular part of the death receptor

=> formation of a big intracellular signaling complex

=> FADD binds

=> binding, dimerization and proteolytic activation of initiator caspase-8

=> cleavage and thereby activation of the effector caspases 3, 6 and 7

=> cleavage of cellular proteins necessary for survival

=> induction of cell death

Caspase-8 can also cleave BH3-only protein Bid => tBid fragments bind to Bcl-2 => release of Bak or Bax à pore formation in the outer mitochondrial membrane => release of Cyt c to the cytosol => activation of intrinsic pathway