Cell cycle control

• Normal physiology requires cells to replicate at defined times and locations so require a start and stop mechanism. Loss of control is implicated in developmental disorders and pathologies.
• Ki-67 is a protein indicative of proliferating cells and is involved in DNA replication.
• Negative and positive feedback mechanisms are used to achieve precise control of regulation.
• The mechanism of cell cycle control uses feedback

  • cell division occurs only after DNA replication
  • cell division occurs once DNA has replicated not allowing for a second (partial) replication
  • allows repair of DNA damage
• 3 main checkpoints during cell cycle

  • end of G1
  • is environment favorable?
  • end of G2
  • is all DNA replicated?
  • Is all DNA damage repaired?
  • M phase
  • are all chromosomes properly attached to mitotic spindle?
• Regulation is largely achieve by:

  • protein phosphorylation (kinases)
  • dephosphorylation (phosphatases)
  • protein degradation (ubiquitinases)
• Protein degradation by ubiquitination

  • addition of ubiquitin tag to protein
  • degradation of tagged protein in proteosome
  • activation by E1
  • transported by E2
  • ligation of ubiquitin by E3
• Regulation by kinases

  • kinases add phosphate groups to proteins at specific residues
  • changes conformation
  • can allow a substrate to interact with an enzyme domain (activate)
  • or hinder access (inactivation)
  • Phosphorylation by kinases needs to be regulated so phosphorylation occurs at the correct time and location (by regulatory proteins e.g. cyclins)
• Cyclins and Cdks - regulator of the kinases
• Cyclins are transcribed in a particular time frame of the cell. The level of protein of a particular cyclin will gradually increase and then rapidly decrease. Cyclin-cdk activity is specific to a point in the cell cycle. Activity increases rapidly despite the level of the regulatory protein (cyclin) increasing gradually.
• Regulation of cyclin:cdk

  • cdks are phosphorylated to inhibit their activity
  • inactive Cyclin-CDK complex
  • a phosphatase activates the cyclin-cdk complex
• • rapid reduction in cyclin:cdk activity is achieved by degradation of cyclin
  • enzymes add a chain of small ubiquitin groups
  • polyubiquitinated proteins are targeted for degradation in the proteosome
• Other proteins can bind to cyclin:cdk complexes and inactivate the complex. Called cyclin-dependent kinase inhibitors (CdkI). In late G1 the CDKIs are degraded by ubiquitination by the enzyme complex SCF.
• • cells only replicate when the external environment sends cues to stimulate cell division/the cell cycle
  • mitogens stimulate the production of cyclins involved in the early stages of the cell cycle
  • cyclins indirectly regulate the transcription of genes involved in processes such as DNA synthesis
• Mitogens - stimulate cell division primarily by overcoming intracellular breaking mechanisms
• Growth factors - stimulate cell growth (increase in size and mass) by promoting the synthesis and inhibiting the degradation of proteins and other macromolecules
• Rb and E2f

  • Rb binds E2F transcription factors
  • phosphorylation of Rb releases E2F transcription factors allowing the interaction with transcription-activating proteins on the promoters of genes required for cell cycle e.g. DNA replication
  • expression of genes required for the cell cycle
• Starting the cell cycle

  • cyclin D phosphorylates Rb to allow the cell to progress through the restriction point
  • prior to the restriction point cell cycle progression is dependent on extracellular signals
  • after the restriction point the cell is committed to the cell cycle - a cell-autonomous process
• Completion of mitosis

  • metaphase to anaphase transition results in the separation of sister chromatids
  • held together by a protein called securin
  • securin is the other main target of the APC/C complex
• Cdk-activating kinase - phosphorylates an activating site in Cdks
• Wee1 kinase - phosphorylates inhibitory sites in Cdks; primarily involved in suppressing Cdk1 activity before mitosis
• Cdc25 phosphatase - removes inhibitory phosphatases from Cdks; three family members (Cdc25A, B, C) in mammals, primarily involved in controlling Cdk1 activation at the onset of mitosis
• Sic1 - suppresses Cdk1 activity in G1; phosphorylation by Cdk1 at the end of G1 triggers its destruction
• p27 - suppresses G1/S-Cdk and S-Cdk activities in G1; helps cells withdraw from cell cycle when they terminally differentiate; phosphorylation by Cdk2 triggers its ubiquitylation by SCF
• p21 - suppresses G1/S-Cdk and S-Cdk activities following DNA damage
• p16 - suppresses G1-Cdk activity in G1; frequently inactivated in cancer
• APC/C - catalyses ubiquitylation of regulatory proteins involved primarily in exit from mitosis; including securin and S- and M- cyclins; regulated by association with activating subunits Cdc20 or Cdh1
• Cdc20 - APC/C-activating subunit in all cells; triggers initial activation of APC/C at metaphase-to-anaphase transition; stimulated by M-Cdk activity
• Cdh1 - APC/C activating subunit that maintains APC/C activity after anaphase and throughout G1; inhibited by Cdk activity
• SCF - catalyses ubiquitylation of regulatory proteins involved in G1 control, including some CKIs; phosphorylation of target protein usually required for this activity.