Exam 3 -BIO457

What are the other checkpoints in the cell cycle (other than G1/S and G2/M)

DNA damage checkpoints (G1/S and G2/M), spindle assembly checkpoint before anaphase, spindle position checkpoints before anaphase, and intra-S phase

What are heterodimeric protein kinases involved in cell cycle composed of?

• regulatory subunits (cyclins), whose concentration cycles in phase with the cell cycle

• catalytic subunit (cyclin dependent kinases/Cdks) that phosphorylate only when cyclin is present;

  
  

  • each Cdk catalytic subunit can associate with different cyclins that determine which proteins are phosphorylated by the CDK-protein complex

Describe G1 cyclin Cdks

target transcription factors(telling cell it needs to wake back up to drive gene expression for stuff needed for cell cycle), replication enzymes/proteins, S-phase cyclin-CDKs/inhibitors

describe S-phase cyclin cdks

target proteins involved in DNA replication

describe mitotic cyclin-cdks

target hundreds of proteins including those that help condense chromatin, cause the breakdown of nuclear envelope, mitotic spindle proteins and numerous other protein kinases

what proteins (class and specific) tag proteins for degradatioin in the cell cycle and what phase do they work in? What guides them to their targets?

ubiqutin protein ligases guided by specifficity factors

• SCF—> induces S-phase in late G1

• APC/C—→ induced anaphase in mitosis

what are the protein phosphatases working in cell cycle?what phases are they involved in?

Cdc14 (late mitosis/cytokinesis) and cdc25 (at major checkpoints)

list all cyclincdks/ubiq protein ligases/phosphatases going around cycle startin at G1

G1 cyclin-cdks prep for S-phase—> G1/S cyclin Cdks and SCF help get into S phase—>S-hpase cyclin cdks activate dna replication—→ mitotic CDKs induce mitosis and SCF heelps get thru G2 to M—> APC/C induces anaphase

what is the fastest vs slowest part of the ceell cycle?

mitsosi is fastest (30 min) and dna replication is slowest (10 hrs)

what can postmitotic cells do if they don’t need to replicate?

• can “exit” the cell cycle and remain for various amounts of time w/o further proliferation

  • exit the cell cycle in G₁ and enter G₀ phase

  • these cells can reenter the cell cycle if stimulated

what experiment discovered factors controlling entry/exit from mitosis and what did they find?

cell-fusion experiments (electrocution to fuse) w/cultured mammalian cells

• interphase cells fused to mitotic cells showed nuclear envellope berakdown and dna condensation indicating some factor in mitotic cell triggered it

• G1 cells fused to S phase cells and exposed to radiolabeled thymidine—> label was incorporated into dna of G1 nucleus to start replicaiton

• BUT when g2 fused to S, no incorporated label (can’t reverse cell ccyle)

how were factors inducing mitosis identifiied? describe everything

oocyte development in frogs

1. oocytes stop in G2 phase for a very long time until stimulated by male

2. progesterone is secreted by ovarian cells, to induce entry into meiosis I

3. arrests again in metaphase II (2-3 = oocyte maturation)

4. egg nucleus is fertilized byu sperm, releasing from metaphase II to finish meiosis

• protein fracttions were isolated from eggs arrested in metaphase II and put into G2 arrested oocytes, causing maturation to eggs, even w/o progesterone

  • found that the factor stimulating this was maturation promoting facot r(MPF)

What does MPF do?

• acts as a kinase to phosphorylate H1 histone, leading to condensation of chromatin to 30 nm fiber

describe the cycling of MPF concentration

• rises and peaks during early mitosis and falls abruptly during anapahse, at which point cyclin B is degraded, to allow cell to finish mitosis

• slowly accumulates during next interphase

what is MPF composed of?

cyclin B with CDK1

how was cyclin B concentration found to be important to cell cycle?

• aftter all mRNAs were degraded with RNase, after which RNase was inacttivated and the egg was fertilized bc it couldn’t translate anthing into proteinn

• when cyclin B was readded, oscillations in MPF activity came back, corresponding to the rise/fall of cyclin B levels—> mitotic events occurred

• when a non-degradable cyclin B was introduced (mutant) to RNasse treated extracts, MPF activty/mitotic structure formed, but later mitosis was blocked

how are cyclins degraded? (describe site targeted and proteins involved)

• all hae a destruction box/D-box sequence near the N-terminus, on which many copies of ubiquitin protein is added to lysines on the C-terminal end of to mark for degradation (polyubiquitination)

• E1/ubiquitin-activating enzyme: binds ubiquitin and gives to E2 (requires atp hydrolysis)

• E2/ubiquitin-conjugating enzyme: recognizes target protein to be degraded at D-box

• E3 or ubiquitin ligase: adds many ubiquitins to lysine w/peptide bond just outside of the D-box toward C-term end

what does APC/C target?

mitotic cyclins and other proteins

what does SCF target for destructtion?

Sic1/p21

what degrades polyubitinylated proteins?

proteasomes

describe the proteasome structure/mechanism

• has caps on either end with inner chamber

• ubiquitin receptors find ubiquitnylated proteins—> protein is deubiquitinated to feed the protein into the chamber

  -        in the chamber the protein is cleaved as it goes through, releasing small peptides on the other end

  
  

what specificity factor targets what protein ligase towards cyclin B? when/how is it activated?

• APC/C is targeted to cyclin B through Cdh1, which remains inactive/phosphorylated by cyclin/Cdk complexes until late interphase

• Cdc14 phosphatase removes P from Cdh1, thus activating APC/C, which mediates polyubiquitination and degradation of cyclin B

  
  

what aa residues does MPF target?

serine

list the targets of MPF

nuclear lamins A/B/C, specific nucleoporins, integral membrane proteins of inner nuclear membrane, chromatin proteins (H1), microtuble associated proteins (MAP), membranes of ER and Golgi complexes, and condensin complexes

describe how MPF targets nuclear lamins and what it causes

• phosphorylates serines in nuclear lamins A, B and C that support the inner nuclear membrane, forming a network of lamin filaments

  • causes depolymerization of lamin filaments leading to membrane breakdown (from teteramer to dimers)

  • is reversible and can get repolymerization of lamins by action of phosphatases

when MPF phosphorylates nucleoporins what happens?

nuclear pores come apart

what happens when MPF phsophorylates integral membrane proteins of inner nuclear membrane

these proteins act like tent stacks for proteins; decreases their affinity for chromatin and helps dissasemble the nuclear envelope (

what happens when MPF phosphorylates H1?

this causes chromatin condesnation to 30 nm fiber

what happens when MPF phosphorylates MAP

this activates MAP to come together and form the spindle fibers/asters for mitosis

what happens when MPF phosphorylates ER/Golgi membranes?

they break into smaller organelles so they can be split between new cells in mitosis

what happens when MPF phosphorylates condensin complexes?

this promotes condesnation of chromatin, allowing condensin to bind it

what is condensin made of?

Smc2/4 and kleisin

what does MPF activity control?

early mitotic events

how is return to interphase conditions triggered in late mitosis?

protein degradation and dephosphoyrlatio

what evidence is there that anaphase is triggered by polyubiq of certain proteins

§  when synthetic polypeptide containing the destruction box of cyclin B is added to untreated egg extract, movement of chromosomes toward spindle poles (proceeding to anaphase) was blocked—→ o   extra destruction-box peptide acts as a substrate for APC/C-directed polyubiquitinylation system which competes w/the normal endogenous target protein (delays/prevents their degradation)

what holds sister chromatids together and what is it made of?

cohesin made of Smc1, Smc3, Scc1 (kleisin) and Scc3

when is cohesin loaded onto chromosomes and by what?

during late G1 to keep sister chromatids together during replication; by Scc2-Sccc4 complex

how is cohesin stabilized?

cohesins are stablized by Sororin

what are cohesins removed by? when does this occur?

by Pds5-Wapl complex to allow chromosomes to separate during anaphase

what activataes Smc3 in cohesin?

cohesion acetyltransferases (CoAT)

when do cohesins hold sister chromatids

during S phase

during G2 what happens to cohesins?

2 molecules of Mei-S332/Shugoshin/PP2A (phosphatase that cleaves off phosphate groups) complex bind to centromere to help protect the last few cohesins around the centromere (resists pulling apart of sister chromatids until anaphase occurs)

how and when are cohesins removed from chromosomes?

phossphorylation/removed by Polo-like kinase 1 (Plk1) and Aurora B kinase during the middle of prophase

what senses tension at the kinetochore before anaphase can occur and what happens if there is not enough tension

Aurora B is positioned at the centromere between two kinetochores

• if  kinetochore is not under tension, it can phosphorylate Ndc80 complex and destabilize kinetochore-microtubule attachments

• allows Ndc80/microtuble to try again to make proper attachement

what is the role of Aurora B in anaphase

acting as a sensor/checkpoint to prevent nondisjunction (incorrect numbers of chromosomes make it into cells during mitosis)

What happens when the kinetochore is under tension that keeps attachments stabilzed for anaphase

If everything is attached/under tensions, Ndc80 can move away from Aurora B and PP1 phosphatase will keep Ndc80 dephosphorylated

how/when are cohesin complexes degraded to allow separation of sister chromatids?

• once spindle fibers have attached to centromere, APC/C, led by specificty factor Cdc20, is targeted to polyub. securin (inhibitor of seaprase)

• securin degradation by proteosomes allows separase to be dephosphorylated

• separase is able to cleave Scc1 (kleisin adjacent in cohesin), breaking cohesin structure and allowing sister chromatids to seaprate

how are proteins involved in chromatin condensation and nuclear envelope breakdown returned to normal?

• Cdc14 phosphatase is activated, allowing it to dephosphorylate/activate Cdh1—> allows cdc20 to target cyclin B of MPF

  • MPF, which phosphorylated a bunch of stuff for mitosis, was inactivated at the beginnin of anaphase, allowing Cdc14 to remove phosphates unopposed

what signals the cell to progress through G1 to S and complete cell sycle/get cells out of G0

growth factor sbinding to receptors

what cyclins are involved in G1? what Cdks do they bind to?

cycliin D’s —> Cdk2 and 4

what cyclins are involved in G1/S? what Cdk do they bind to?

cyclin E to cdk2

what cyclins are involved in S-phase? what Cdk do they bind to? which one is driving S-phase

cyclin E and cyclin A—> to Cdk2; cyclin A-Cdk2 is driving S-phase

describe the concentration of Sphase cyclins in the cell cycel

they are synthesized at the same time as G1 cyclins but remain high through Sphase and don’t decline till early mitosis

what cyclins are involved in M-phase? what Cdk do they bind to?

cyclin A and b—> bind cdk1

what do growth factors do to trigger cell cycle?

they induce synthesis fo cyclins E and E, Cdk2,4,and6 (induce early response/delayed response genes)

describe induction of early response genes

• genes of cFos an cJun transcription factors are induced—> these proteins form heterodimers to bind DNA at specfic promoters/networks (including that of the delayed response genes)

• isn’t blocked by inhibitors of proteins synthesis bc cell always keeps a little bit of cFos/cJun around in the cell

describe where discovery of cFos and cJun come from and what kind of protein they are

these are leucine-zipper proteins TFs

• retroviruses carrry oncogenic, unregulated forms of these factors (vFos and vJun)—> if these are expressed in normal cells they turn malignant

describe concentratioin of early response mRNAs when growth factors are introduced

• peak in 1st 30 min after GFs are added and after drop to lower level that is mainted as long as GFs are present

describe the delayed response genes

these are for S-phase—> encode TFs, such as E2F, which stimulates transcription of cyclin E, cyclin A, Cdk2, PCNA, DNA pol alpha/sigma/episilon, RPA, MCM, and Cdc25A

describe regulation of E2F

it binds to other proteins that inhbiti it —> prevents transcription

1) RB protein – encoded by tumor suppressor gene

2) p107 – same

3) p130- same

what targets the Rb protein for phosphorylation and why? what keeps Rb phosphorylated and whe?

• by G1 cyclin-CDK complexes (cyclinD and CDK4/6) to release E2F, allowing DNA transcriptioin of cyclin E/A to get into S phase

• S-phase and mitotic cyclin-CDKs during S-phase through M

what proteins control activity of S-phase complexes?

• CDK inhibitory proteins (CKIs)

  • p21, p27, p57

how is p21 or other CDK inhibitory proteins degraded?

hyperphosphorylation by cyclin E-CDK2 to cause conformational cchange/fall off and marked for degradation by by SCF

what does p21 do?

inhibits all cyclin-cdk complexes

when do cyclin B levels start rising and when do they associate with Cdk1

late S-phase; G2

what activates Cdk1 and how? when?

Cdc25C phosphatase by dephosphorylation at G2/M transition

what activates Cdk2 and how?

Cdc25A phosphatase by dephos at G1/S transition

descibe how cdc25a/c do differential phosphorylation

§  Cdc25A targets CDK2 while Cdc25C targets CDK1

what inhibits Cdk1 and how?

Wee1 kinase through phosphorylation of threonine 14 and tyrosine 15

what TF do cFos and cJun make up?

AP-1

describe the intra-S phase checkpoint (purpose, how is it regulated)

• makes sure all DNA is replicated before entering mitosis

• Cdc25A/C are inhibited from activating CDK2/1

  • ATR kinase phosphorylates Chk1/Chk2 which phosphorylates/inactivates Cdc25 A/C (when fork stalling or ssDNA is detected)

describe the spindle assembly checkpoint (purpose, what

• prevents nondisjunction (chromsomes don’ t split evenly)

• to sense tension: Knl1 (kinetochore component) is phosphorylated on tyrosines by Mps1 kinase—> phosphotyrosines act as docking sites for Bub1 kinase-Bub3 and BubR1 (which sense the tension)

• Mad1-Mad2((2 of each) complex (mitotic arrest deficiency) is recruited to bind Bub1-Bub3/Mad3 in cytoplasm,—> forms MCC (mitotic checkpoint complex)

• converts to more Mad2-O into closed form Mad2-C, which is active, in the cytoplasm

• Mad2-C binds Cdc20- APC/C to inhibit its function —> securin can’t be degraded—> separase can’t function to cleave off cohesin and allow anaphase

• PP1 (protein phosphatatse 1) dephosphorylates Knl1 once kinetochores have joined the microtubles —> disassembles MCC

• p31 helps release Cdc20 from APC/C and disassmble MCC

describe the spindle position checlpoint

• checks to make sure they are 180 degrees from each other for proper cytokinesis

• depends on mitotic exit network proteins (Tem1, located at the spindle pole bodies, acts as a GTPase that is activated when chromosomes start moving toward the poles)

• Tem1 promotes release of Cdc14 (dephos of Cdh1 to allow it to join MPF and degrade MPF) to promote inactivation of mitotic cyclin-cdks and completion of the cell cycle

describe the DNA damage checkpoints (purpose, targets)

• prevents replication and cell division when DNA damage is detected

• stops in G1 to prevent copying of incorrect bases or G2 to repair double stranded breaks

• ATM/ATR kinases activated and phosphorylate (to activate)…

a)   Chk1 and Chk—>  phosphorylate Cdc25C and Cdc25A during intra-S checkpoint

·       phosphorylation of these phosphatases by marks them for polyub and degradation (SCF for Cdc25A)

·       without these phosphatases CDK1/2 can’t be dephosphorylated and cell will be arrested

 

b) p53

·       helps arrest cells with damaged DNA in G1 and G2 until all damage is repaired, after which p53 and p21 levels fall

·     transcription factor whose activation leads to transcription of specific genes that help repair damage

·       upregulates p21 production which binds to and inhibits all CDK-cyclin complexes (halting cell cycle)

·       if the dna damage can’t be fixed—> apoptosis is triggered

what does stalled replication trigger? what checkpoint is this?

intra-s checkpoint—> ATR—> Chk1—> Cdc25C

what doe DNA damage trigger in S-phase?

ATM/R senses damage—>activates p53—>p21( all cyclin-CDK complexes are inhbitied)

what does correct spindle-position tirgger?

mitotic exit network’s Tem1 GTPase is activated—> Cdc14 released—> dephosphorylates Cdh1—> joins with APC/C to degrade MPF—> end of cycle

what is an oncogene?

encode proteins able to transform cells in culture or induce cancer in animals; almost all are derivatives of normal cellular genes

what are oncoproteins? how to they compare to proteins from proto-oncogenes?

protein products encoded by oncogenes and they act along normal cellular growth-controlling pathways

o   some oncoproteins are mutant forms of the normal product (changes structure)

o   sometimes the same, but it causes cancer bc it is normally absent or is being overexpressed

o   mutation creating the oncogene changes transcriptional control rather than protein structure

what are proto-oncogenes?

aka tumor suprressors; genes that are very important fornormal celullar processes and create oncogenes when altered

how are oncogenes /oncoproteins designated?

with a 3 letter code where the gene is italicized and the proteins are normal

list the 7 types of oncoproteins

1) extracellular signalling molecules

2) receptor

  3) intracellular signal transducers/second signal messengers

4) transcription factors

5) cell cycle control proteins

6) apoptotic proteins

7)DNA repair proteins

what are two common extracelllular signalling molcules that induce the cell to grow?

• EGF (epidermal growth factor)

• PDGF( platelet-derived growth factor)

what oncoprotein encodes a type of PDG