Cell Cycle

  1. Mammalian cell cycle
    • G1 - cell growth, increase in phase/size; centrosome replication in late G1 phase
    • S - DNA rep
    • G2 - DNA exist as chromatid, centrosome separation
    • Prophase - centrosome migration; choromsome condensation; appear as long threads
    • Prometaphase - spindle formation; nuclear envelope fragmentation; chromsome condensation completed and held together at centromeres by spindle microtubules
    • Metaphase - chromosome alignment
    • Anaphase - chromosome separation
    • Telophase/cytokinesis - nuclear membranes reformation; chromsome decondensation; spindle diassembly
    • Cytokinesis - cyto plasmic division by the contractile ring
  2. MPF
    mitotic cyclin (cyclin B) + mitotic cdk (cdk1 = cdc2)
  3. cdk1 or cdc2
    • cdk1 - cyclin dependent kinase, also
    • cdc2 - cell division cycle
    • ser/thr protein kinase
    • no acvitivy without cyclin
  4. Regulation of Cyclin Dependent Kinases
    • Cyclin association/destruction
    • Activating phosphorylation events on cdk
    • Inhibitory phosphorylation events on cdk
    • Cdk inhibitors (CKIs)
  5. Cdc14
    • a phosphatase that removes phosphate from cdh1, activating ch1 so it binds to APC/C and activate APC/C
    • only actie in late anaphase
  6. Anaphase promoting comlex/cyclosome (APC/C)
    • APC/C binds cdc 20 to ub securin and activate separase to separate sister chromatids
    • G1 cyclin phosphorylates cdh1, deactivates it, cdh1 cannot bind APC/C, APC/C remains inactive
    • cdc14 phosphatase (only active in late anaphase) dephosphorylates cdh1 and ch1 binds to APC/C - activating it
    • cdc20 and APC interaction is before cdh1; cdc20 is inhibited until all chromosomes are attached and aligned
    • cdh1 activity is inhibited until the sister chromatids are separate
    • functions as a checkpoint to ensure that chromosomes remain condensed and nuclear envelope does not assemble until chromosomes are properly agined
  7. wee1
    • excess of wee1 leads to elongated cells, incrased G2
    • deicit of Wee1 leads to small cells, decreased g2
    • wee 1 phosphorylates inactive MPF on Y15
    • leads to CAK to phosphorylate (CDK activating kinase) on T161
    • cdc25 dephosphorylates on Y15 will activate cdc2 (cdk1) makes active MPF
  8. cdc25
    • in the budding yeast
    • activates CDK, final step to make active CDK, dephosphorylate at Y15
  9. human cdk2
    high activity: has Thr160 phosphorylated - substrate binding site
  10. MPF substrates
    • Nuclear lamins
    • phosphorylation of Lamin tetramers by MPF initiates disassembly
    • Proteins invovled in chromosome condensation, spindle assebmly, chromosome alignment
  11. nuclear enelope breakdown and reformation
    • Interphase - MPF leads to phosphorylation of lamins - disassociation
    • Prophase - nuclear envelope fragment, after prophase Lamins are dephosphorylated
    • Early Telophase - by then, lamins are dephosphorylated and reassembbly
    • Late Telophase - by then, fusion of nuclear envelope fragments
    • After telophase - fusion of enveloped chromosomes
  12. p97
    mediates homotypic vesicle fusion
  13. cohesion
    • smc2, smc4 + kleisin
    • SMC protines: sturcutral maintenance of chromosome proteins
    • Function: link daughter chromosomes together
    • From G2(after replication of chromatin) to during metaphase, cohesion concentrates around centromere, concentration regulated by phosphorylation
  14. cdc20
    • bidngs to APC/C, acivating it, so that it polyubiquitinates securin for degradation
    • degradation of securin leads to release of spearase from separase + securin complex, separase is activated and cleaves kleisin (cohesion cleaved)
    • sister chromatids separate
  15. Cyclin Destruction Box
    • 9 amino aacids, directs polyubiquitination on lysine, target for destructino by proteasome.
    • Entry into mitosis requires accumulation of cyclin B
    • Exit from mitosis requires degradation of cyclin B
  16. cdc28
    • the only cdk in budding yeast cells - a cell division kinase
    • cdc28=cdc2 in fission yeast = cdk1 in mammals
  17. SPF
    • s-phase promotin factor
    • Budding Yeast: cyclin 1, 2 (G1 cyclin) + CDK (cdc28)
  18. G1 cyclin yeast, mitotic cyclin in yeast, mitotic cyclin in mammals
    • cyclin 1 and cyclin 2 (cln1, cln2)
    • clb1, clb2
    • cyclin B
  19. Sic 1
    • S-phase inhibitor in budding yeast
    • binds to SPF and inhibits
  20. 4 mechanism of regulating CDK
    • cyclin association/destruction
    • activating phosphorylation events on cdk
    • inhibitory phosphorylation events on cdk
    • cdk inhibitors (CKIs)
    • Note: cdk protein levels remain relatively constant throughout the cell cycle
  21. degradation of the S-phase inhibitor
    • G1/S trasition
    • sic1 binds to Sphase cyclin (clb 5,6) and cdc28(cdk) and inhibits SPF (Sphase cyclin + cdc8)
    • G1 cyclin (cln1) and cdc28 (cdk) phosphorylate sic 1
    • leads to polyubiquitination of phosphorylated sic 1 via SCF (Ub E3 ligase) and proteasomal degradation
    • required for G1/S transition - lead to DNA replication
  22. SCF
    • Skp1/Cul1/F-box protein = E3 Ub Ligase
    • mediates G1 cyclin regulators degradation and activates G1 cdk, required for G1/S transition
  23. ORC
    origin recognition complex, bind to origins of replication
  24. cdc6
    DNA helicase loading factor, increased in early G1, bind to Oc
  25. MCM
    DNA helicase
  26. pre- RC
    • pre replication complex
    • ORC + cdc6 + MDM
  27. initiation of DNA replication
    • ORCs bind to origins of replication
    • Cdc6 (DNA helicase loading factor) level is increased during early G1 and binds to ORC
    • ORC, cdc6, and Mcm (DNA helicase) form a complex called pre-RC (replication complex)
    • accumulated s-cdk phosphorylates cdc6
    • cdc45 binds to Mcm - activates Mcm, unwinding of parental DNA strand leads to release of cdc6-p and ctd1-p
    • release of cdc 6-p lead to degradation, allows: Mcm to move and assembly of replication fork (RPA, DNA polymerase)
    • cdc6 is inhibited by phosphorylation, degradation and nuclear export until M-cdk is inactivated at the end of mitosis
  28. experiment: microinjection of cyclin D antibodies
    • BrdU is a synthetic nucleoside that is an analogue of thymidine - can be incorporated into DNA during replication
    • G0 cells were treated with growth factor and either injected with antibody or anti-cyclin D antibody, then added brdU, wait 16 hours
    • Injection of control antibody: BrdU positive cells - rep occured, BrdU incorporated into DNA
    • Injection of anti-cyclin D antibody: Cylin D bound to antibody, acnnot get in S phase, no DNA rep, BrdU-negative cells (not incorporated into DNA)
    • Conclusion: Cyclin D is important for DNA replication
  29. Rb
    • Retinoblastoma protein - a tmor suppressor gene (inactivated in most human cancer cells)
    • Mid G1: Rb binds E2F, inhibiting E2F
    • Mid G1 cyclinD + Cdk4/6 (induced by growthfactor) and Latae G1 Cyclin E + Cdk2 both phosphorylate Rb
    • Rb-p releases E2F, E2F is activiated - lead to positive feedback, further phosphorylation by S-cdks
    • S cdks accumulate activated E2F lead to DNA rep and G1/S transition
  30. p53
    • Detects damage to DNA throughout the cell cycle
    • a tumor suppressor and trasncription factor, stabilized p53 activates transcription fo p21 CIP (a CDK inhibitor)
    • p21 gene is translated into CDK inhibitor protein
    • Mdm2 is aubiquitin ligase - p53 negatively regulated by mdm 3
  31. Spindle Checkpoints
    • kinetochore-mediated: cdc20
    • MTOC-mediated: mad2
    • ensures all cell cycle is only one directional
  32. mad 1 and mad 2
    • mad 1 binds to a kinetochore that is not connected to microtubule
    • mad 2 can bind to mad 1, from open conformation to closed conformation
    • another mad 2 can bind the mad1/close mad2 and become closed
    • the newly closed mad 2 binds cdc20, inhibiting cdc, more open mad 2 bind closed mad2/cdc20, and bind other cdc20 to inhibit them
    • inactiviation of cdc20 inhibits cdc20-APC interaction, so secruin is not degraded
    • spingle-assembly checkpoint'
    • cell cycel is inhibited until all the chromosomes are aligned and all kinetochores were attached by MT, mad1/2 will deatch, and bind p31
  33. spindle assembly checkpoint inactiviation
    • when the MT binds to kinetochores, mad1/close mad2 tetramers are released from kinetochore and p31 binds
    • p31 also binds closed form of mad 2 and frees cdc20, closed mad2 becomes opened mad2, and free p31 floating around
    • cdc20 will bind APC/C
    • lead to secruin degradation
    • entry into anaphase
  34. Spindle-position checkpoint
    • SPB: spindle polary body, first in the mother, entrance into the bud trigger TEM1-GDP to be activated, Cdc14 was originally stored in the nucelolus, will be distributed, to that stored (inactive) cdc14 becomes active (spread out)
    • TEM1-GTP is the active conformation
    • if SPB remains in mother cell, TEM remains inactivated (GDP bound), cdc14 remains in nucleus, leads to mitotic arrest
    • TEM1-GEF only in bud; Kin4 (TEM-GAP activator in cortext of mother cell)
    • Note: cdc14 phosphatase activity is required for exit from mitosis
  35. Tem1
    small GTPase that activates MAPK pathway, important for proper chromosome segregation/budding
Card Set
Cell Cycle
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