Meeting 28

  1. APC/C (Anaphase promoting complex)
    a ubiquitin-protein ligase polyubiquitinylates SECURIN, which is then degreaded by proteosomes

    • -SECURIN: normally acts to prevent degredation of proteins that link sister chromatids at their centromeres
    • -when its destroyed, sister chromatids can separate, starting ANAPHASE

    -APC/C also causes degredation of cylics late in anaphase...causes Telophase and ending of mitotic shit
  2. MPF (mitosis promoting factor)
    Image Upload 1
    • MPF and cyclin work together to start mitosis
    • cyclin is a key component of MPF; it's the component that cycles...relies on mRNA translations and subsequently degredation when the cell wants mitosis to end
  3. cyclin-CDK complexes
    • so there are these heterodimers made up of
    • 1) cyclin: the regulatory subunits whose concentrations fluctuate during the cell cycle
    • 2) CDKs (cyclin-dependent kinases) the catalytic subunits that express enzymatic activity ONLY when associated with a cyclin
    • -their levels don't fluctuate like those of cyclin do

    TOGETHER: these subunits activate or inhibit proteins involved in cell-cycle progression by phosphorylating them at specific regulatory sites

    -peaks of cyclin PRECEED cell division; cell synthesizes a lot of cyclin 1ST --> then division happens
  4. CDC2 (or 28)
    • protein kinase; a CDK
    • -if it's recessive in the cell: mitosis/division never occurs; cell stays in G2 phase and keeps elongating
    • -if it's dominant in the cell (forced cdc2 activity: the cell is forced to divide before its grown to its normal WT size
  5. G1 cyclin-CDKs
    • -prepare cells for S phase
    • -phosphorylate Tx factors required for chromosome replication (ex. enzymes that make DNA pol, or S-phase cyclins necessary for initiating DNA replication)
  6. SCF upiquitin-protein ligase
    • -controls passage through cell cycle transitions
    • 1) G1 --> S (induces S phase)
    • 2) metaphase --> anaphase
    • 3) anaphase --> telophase/cytokinesis

    • -regulate MPF activity & cohesion between centromeres of sister chromatids
    • -such transitions are created by the DEGREDATION of proteins, which is irreversable, SCF's force the cells to pass through the cell cycle unidirectionally
  7. S-phase cyclin CDKs
    • -activate DNA replication origins
    • -made in late G1, then immediately bound by inhibitors
    • -phosphorylated by G1 cyclin-CDKs
  8. Mitotic cyclin-CDKs
    • -activate early steps in mitosis
    • -made in S phase and G2, not activated till DNA synthesis is complete
    • -once phosphorylated, they activate proteins (100s) that: promote chromosome condensation
  9. checkpoint mechanisms
    • checkpoints ensure that cell-cycle transitions aren't initiated until the previous process upon which they depend is complete
    • -include: S-phase, start of mitosis, separation of daughter chromosomes @ anaphase, & start of telophase/cytokinesis
    • -function by controlling cyclin-CDKs (MPFs) by:

    • 1) regulating the concentrations of cyclins
    • 2) phosphorylating CDKs
    • 3) making/controling the presence of CKIs (CDK inhibitors)
    • 4) regulation of APC/C complex
  10. APC/C degrades cyclin
    • Cdh1: a specificity factor that BINDS to APC/C and directs it to degrade cyclins WHEN PHOSPHORYLATED
    • -so it's BOUND during late anaphase/telo/cytokinesis, b/c that's when low levels of cyclin are found (cyclin promotes entry to mitosis, those are the final stages of mitosis, therefore it makes sense that this is when APC/C needs to degrade cyclin so there isn't an arrest in mitosis)

    -Cdc14 (a phosphatase) REMOVES the phosphate from Cdh1, releasing it from the APC/C complex, allowing the level of cyclin to once again rise (beginning mostly at G1) and prepare the cell for mitosis
  11. cyclin-CDK complex is regulated by phosphorylation/dephosphorylation

    Image Upload 2
    • holy poop
    • -Cdc25: phosphatase -Wee1: tyrosine kinase
    • -they have opposite effects on the MPF

    • -remember: cells needs high levels of cyclin to enter mitosis/divide
    • -Wee1 phosphorylates CDK (+ another kinase); in EXCESS, makes the cell divide/enter mitosis before its ready/reached the proper lenght
    • -Cdc25 is a phosphatase so it DEphosphorylates CDK attached to cyclin (MPF), meaning the two can never innitiate mitosis: cell just grows, doesn't divide

    -I don't really know how though, basically affects substrate binding site...what's the substrate?
  12. Cyclin D
    experiment: injected cells arrested in G0 phase with a) BrdU (thymidine analog, so would flouresce when incorporated into DNA showing S phase) and b) antibody to cylin D

    results: cyclin D is required for transition from G1 to S phase; required to pass the restriction point, but once cells have passed the restriction point, they don't require cyclin D to enter S phase
  13. Transition from G1 --> S phase: Cyclin D-CDK4/6 phosphorylates Rb (inhibiting it)
    the players in this situation are:

    - CylinD + CDK 4/6: when signaled by mitogens, it phosphorylates Rb (inactive form, NOT blocking E2F)

    • - Rb (Retinoblastoma protein): initially inhibits E2F actvity
    • -active form (blocking E2F): no phosphates
    • -inactive form (E2F is free!): phosphorylated

    - E2F: Tx factor that stimulates Tx of genes for cyclinE, CDK2, and E2F itself (autostimulation!)

    • - Cyclin E + CDK 2: further phosphorylates Eb (positive feedback loop!)
    • -results in a rapid rise in expression/activity of E2F and Cyclin E + CDK 2 itself

  14. Another Method for G1 --> S Phase: phosphorylation of CKIs (Sic1) by G1 cyclin-CDK allow S-phase cyclin-CDKs to trigger DNA synthesis
    • - Sic1 (in yeast; CKIs, CDK inhibitor): in its active state, no phosphates, inhibits S-phase cyclin-CDK (aka DNA rep.)
    • -when phosphorylated, is ubiquitinized by SCF and then degraded by proteosomes

    - G1 cyclin-CDK: PHOSPHORYLATES Sic1 (in late G1)

    • - S-phase cyclin-CDKs: accumulate in G1 but are inhibited by Sic1 (prevents DNA rep. until cell is prepared)
    • - once Sic1 has been degraded though, these complexes can trigger DNA synthesis by themselves phosphorylating preinitiation complexes
  15. p53: Tumor Suppressor in DNA damage-induced cell cycle control
    Image Upload 3
    • -expression of p21 CIP (is a CKI; will block progression of cell cycle)
    • -kind of protein you want to activate if you want to check that everything’s alright

    • p53:
    • -activated in response to some type of damage (DNA mutation)
    • -in HEALTHY cells w/ no DNA damage, it’s regulated by only CDK-cyclin
    • -produced, but never reaches really high levels (constitutively ubiquitinated)
    • -if DNA is damaged; complex is activated, aka remains phosphorylated & isn’t degraded which phosphorylates p21 CIP (ACTIVATING IT), thereby blocking progression of the cell cycle
    • -if the damage is so bad that it can’t be repaired, p53 will eventually trigger apoptosis
    • - becomes mutated in cancer cells; cells don’t die and mutated ones proliferate

    • - ATM/R appears to dephosphorylate p53
    • - Mdm2 tags dephosphorylated p53 with ubiquitin for proteosome degredation
Card Set
Meeting 28
Eukaryotic Cell Cycle