Chapter 21 Regulating the eukaryotic cell cycle Definitions Facts

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Chapter 21 Regulating the eukaryotic cell cycle Definitions Facts Powered By Docstoc
					                                                                           Definitions

                                                           • Cell cycle = an ordered series of
         Chapter 21: Regulating the                          macromolecular events
           eukaryotic cell cycle                           • Cell division = production of two daughters

                       853-6; 881-90
                      Review: 839-840




                                Facts                                   Control factors
   • Regulation of cell cycle is critical for              • Heterodimeric protein kinases
     normal development
                                                             – Cyclin = regulatory subunit
   • Loss of control → cancer
   • Two key events:                                         – Cyclin-dependent kinase = catalytic subunit
      – Replication: controlled by regulating the timing        • Regulate the activities of proteins involved in
        of DNA replication and mitosis                            DNA replication and mitosis
      – Segregation                                             • Phosphorylation at specific regulatory sites
                                                                • Activating or inhibiting




   Overview of cell cycle: Fig. 21-1                       Review of mitosis: Fig. 20.29-20.30
• 4 major phases
   – G1: preparing for DNA
     synthesis and chromosome
     replication
   – S (DNA synthesis)
   – G2: preparing for mitosis
   – M: mitosis
• Cell cycle
   – Yeast: 30 min
   – Human: 24 hrs
• When exit mitosis:
   – G0 = no more division
   – Next G 1 = continue cell
     division




                                                                                                                    1
                     Interphase                                             Prophase
                                                            • Centrosome migration
• DNA and chromosome replication                            • Chromosomes begin to condense
• Made two centrosomes                                      • Centrosomes move to opposite poles




                      Prophase                                          Prometaphase
      • Centrosome migration
                                                        • Spindle formation
      • Chromosomes begin to condense
                                                        • Nuclear envelop fragments = lost
      • Centrosomes move to opposite poles
                                                        • Spindles form
                                                        • Sister chromatids held at centromeres & guided to
                                                          opposite side
                                                        • Kinetochores attach chromosomes to spindle
                                                          microtubules




                  Prometaphase                                             Metaphase
  • Spindle formation
  • Nuclear envelop fragments = lost
                                                            • Chromosome alignment
  • Spindles form
  • Sister chromatids held at centromeres & guided to
    opposite side
  • Kinetochores attach chromosomes to spindle
    microtubules




                                                                                                              2
                Metaphase                                            Anaphase
  • Chromosome alignment                                   • Chromosome separation
                                                           • Sister chromatids are separated into
                                                             independent chromosomes
                                                           • Each chromosome is attached to a
                                                             kinetochore microtubule
                                                           • Chromosomes move toward poles
                                                           • Start of cytokinesis




                 Anaphase                                  Telophase and cytokinesis
• Chromosome separation                              •   Nuclear membrane reform
• Sister chromatids (held together by securin) are   •   Chromosomes decondense
  separated into independent chromosomes
                                                     •   Spindle disappears
• Each chromosome is attached to a kinetochore
  microtubule                                        •   Cell cleavage
• Chromosomes move toward poles
• Start of cytokinesis




                Interphase                                              Movie
       • Enter G1 phase                                   • a20-03-mitosis.swf




                                                                                                    3
             Factors in cell cycle                          Three major classes of cyclin-CDK
       • Cyclins                                             complexes that control cell cycle
          – Regulatory subunits of the heterodimeric
            protein kinases that control cell-cycle
            events
          – Increase or decrease through cell cycle
       • Cyclin-dependent kinases (CDK)
          – Catalytic subunits                                              mitotic
          – No kinase activity until associated with a                                               G1
            cyclin
          – Can associate with different cyclins
                                                                                                S




             Cell cycle: Fig. 21.2                         Early G1: Prepare the cell for S-phase
                                                             • Cell stimulated to replicate (1)
                                                             • DNA replication enzymes → pre-initiation
                                                               complex
                                                             • G1 cyclin-CDK complexes expressed
                                                             • Activate transcriptional factors
                                                             • Transcribe genes (3)
                                                               – Enzymes require for DNA synthesis
                                                               – S-phase cyclins
                                                               – CDKs




   Late G1                                                                            S
                                                                  • DNA replication (6)
                                                                  • Each chromosome only replicate
                                                                    once
• Activity of S-phase cyclin-CDK complexes is initially
  held in check by inhibitors (4)                                    – Phoshorylation of DNA replication
                                                                       enzymes prevents reassemble
• In late G1-phase, G1-phase cyclin-CDK complexes
  induce degradation of S-phase inhibitors (marked by
  phosphorylation & polyubiquination; send for
  proteasomal degradation by SCF ligase) (5)
• S-phase cyclin-CDK complexes are activated →
  phosphorylate/activate DNA pre-replication enzymes (on
  the origin of replication)




                                                                                                           4
                    S and G2                                             Mitosis
• Mitotic cyclin-CDK complexes synthesized          • Anaphase promoting complex (APC, 8 and 9):
• Held in check                                       – Multisubunit ubiquitin ligase
                                                      – Polyubiquitinates key regulatory enzymes (securin)
• Activate (dephosphorylation of inhibitory site)       for degradation
  of mitotic cyclin-CDK complexes → promote           – Securin: inhibits degradation of cross-linking
  proteins involved in (7):                             proteins between sister chromatids
  – Chromosome condensation and alignment             – Degradation of securin → degradation of cross-
                                                        linking proteins → free sister chromatids →
  – Retraction of nuclear envelope
                                                        anaphase
  – Assembly of spindle




                     Mitosis                                              Movie
• Late in Anaphase, APC (9) directs
  polyubiquination/proteasomal degradation of             • a21-01-cell_cycle_control.swf
  mitotic cyclins → inactivation of mitotic
  CDKs → allow phosphatases to act on
  (dephosphorylate):
   – Chromosome deconsation
   – Nuclear envelop reforms
   – Cytoplasm divides (cytokinesis) →
     two daughter cells




                   Cell cycle                            Mammalian cells: Fig. 21-28
      • Passage through cell-cycle transitions is
        irreversible
      • Regulated by degradation of proteins =
        irreversible process
      • Cell cycle is regulated by synthesis &
        activity of cyclin-CDK complexes
      • Activity = discussed above
      • Initiate cell cycle with extracellular
        growth factors (mitogens) → synthesis
        of G1 cyclin-CDK complexes




                                                                                                             5
         Mammalian cells: Fig. 21-28                                                       Return to cell cycle: G0 to G1
 • G1 cyclin-CDK complexes                                                           • Adding mitogens (growth factors) to from G0 to
    – Cyclin D-CDK4                                                                   G1
    – Cyclin D-CDK6                                                                    – Activate early response genes
    – Cyclin E-CDK2                                                                        • Transcriptional factors for these genes are present; only
                                                                                             have to activate
 • S cyclin-CDK complexes
                                                                                           • Make transcriptional factors for delayed-response genes
    – Cyclin A-CDK2
                                                                                       – Delayed-response genes encode:
 • G2                                                                                      • More transcriptional factors
    – Cyclin A-CDK1                                                                        • Cyclins
    – Cyclin B-CDK2                                                                        • CDKs
    – All B cyclins contain a conserved
      destruction box recognized by APC ligase




                Example: E2F and Rb                                                          Tumor suppressor gene Rb
    • E2F factors:                                                                     • Phosphorylation of Rb --> no more E2F/Rb complex
         – Encoded by delayed-response genes                                             --> E2F activates transcription of genes needed for
         – Autoregulate                                                                  S-phase
         – Activate
            • Proteins involved DNA synthesis
            • Transcription of late-G1 cyclins (cyclin D), S-phase cyclins (cyclin
              A), and S-phase CDK (CDK2)
    • E2F + Rb → repressing of E2F activity (Fig. 21-30)                                                                            Mutation of Rb →
                                                                                                                                    Continuous cell cyle
                                                                                                                                    → Tumor


                                                    X
Repression of
E2F activity =
no cell cycle




                  Checkpoints: Why?                                                             Checkpoints: Fig. 21-32
 • Catatrophic genetic damage if cell progress to next phase
   before ready
    – Production of extra chromosome in meiosis → down syndrome
 • Checkpoints = control mechanisms to ensure that:
    – Chromosomes are intact before entering next stage
    – Each stage of the cell cycle is completed before entering next
      stage
 • Four types of checkpoints:
    –   Unreplicated DNA
    –   Spindle assembly
    –   Chromosome-segregation
    –   DNA-damage




                                                                                                                                                           6
                     Checkpoint 1                                  Checkpoint 2
                                                                 • Spindle-assembly checkpoint
• Unreplicated-DNA checkpoint
   – Recognition of unreplicated DNA and inhibition of mitotic   • Improper assembly of mitotic
     promoting factors (MPF) → do not enter mitosis                spindles prevents initiation of
• ATR and Chk1 protein kinase                                      anaphase
• Cells that failed to replicate all their chromosome do         • Mad2 associates with a loose
  not enter mitosis                                                kinochore → inhibits Cdc20
• Association of ATR to replication fork activates its             Mad2 + Cdc20 → no entry to
  kinase activity → phosphorylates Chk1 →
  inactivates Cdc25 → no entery                                    anaphase
• Under normal conditions,                                       • Normally, Cdc20 (target APC to
  Cdc25 activate CDK                                               securin)
                                                                     – APC → polyubiquination/proteasomal
                                                                       degradation of securin → cell enters
                                                                       anaphase




   Checkpoint 3                                                                          Checkpoint 4
• Chromosome segregation check-                                  • DNA-damaged
  point                                                            checkpoint
• Tem1 = GTPase switch protein                                   • Blocks progression through
• Normal Cdc14 is kept inactive                                    cell cycle until damage is
• During anaphase, spindles position                               repaired
  Tem1 in contact with GEF
  (guanosine nucleotide exchange                                 • Arrest in G1 and S prevents
  factor) (Fig. 23-33) → activation of                             replication of damage bases
  Tem1 → phosphorylates inhibitors                               • Arrest in G2 allows double-
  of Cdc14 → Inhibitors released →                                 stranded breaks to be
  Cdc14 available → progression to                                 repaired before mitosis
  telophase & cytokinesis




                     Checkpoint 4                                Well-known Checkpoint 4
                                                                 • p53 and p21CIP (cyclin inhibitor protein)
 • ATM and Chk2 = tumor suppressor genes
                                                                 • p53:
 • DNA damage → activates ATM (kinase) →                             –   Transcriptional factor
   phosphorylates Chk2 = activates Chk2 kinase                       –   Extremely unstable thus normal no activity
   activity → phosphorylates Cdc25A = marking                        –   Arrests cell cycle in G 1 and G2
   for ubiquination → cell arrest                                    –   Activates genes that help the cell cope with DNA damage
                                                                         (Severe damage, p53 will activate apoptosis = cell death)
 • Normally, Cdc25A activates CDK2→ entry to
   S-phase                                                       • p21CIP:
                                                                     – Binds and inhibits cyclin-CDK complexes
 • Mutation of ATM                                                   – Cell arrested in G1 or G 2 for repair
   and Chk2 → no                                                 • DNA damage → Binding of ATM or ATR to p53
   check, no cell arrest                                           prevents rapid degradation of p53 → activate genes
   → cancer                                                        (p21CIP) to help cope with cell damage
                                                                 • Mutations of p53, p21CIP, and ATM/R → cancer




                                                                                                                                     7
      Homework
• 1, 12 b (only Rb), 13
• Analyze the data




                          8