BIOL 2020 Lecture Notes - Lecture 9: Cyclin-Dependent Kinase, Protein Synthesis Inhibitor, The Sea Urchins
Cell Reproduction
November 18 – Dec 2, 2015
Read pg. 611-614
• Experimental pathways
• Discovery and characterization of MPF
*MISSING NOTE FROM NOV 18
Summary: Maturation Promoting Factor Discovery
• Cell fusion experiments
• Amphibian oocytes – Progesterone *relate back to signalling
• Maturation – MPF
• Cycling of MPF – frog embryos
o MPF activity goes up or down with cell cycles
• Cycling of MPF – mammalian cells
• Sea urchins – protein synthesis inhibitor
o If a protein synthesis inhibitor is placed within embryo, it will not divide (even in
desirable environments) – suggests that protein synthesis is required for the regulation
of the cell cycle
Identification of Cyclin:
• Discovered by Tim Hunt using sea urchins
• The sea urchins were incubated with [35S] methionine that will then be incorportated within the
embryos – looks like normal amino acid
• They organisms were then killed and the samples were ran over SDS polyacrylamide gel for
electrophoresis
• Using autoradiography on the gels, they could localize on the gels where the radioactive proteins
are
• In protein A - The division of the cells increased and then subsequently decreased over time but
overall increases as time goes on
• In protein B – the division of cells followed similar pattern to protein A ***Cyclin increases or
decreases with cell cycle
• Another protein continuously increased
• Cyclin –
• Suggests that cell degrades cyclin for the cell to stop reproducing = controlled proteolysis
• Disappears and reappears similar to MPF
Cyclin A mRNA Injection – Xenopus Oocytes
• Experiment by Joan Rudderman
• Had a clone gene that coded for cyclin (function uncertain)
• The gene was then transcribed (in vitro) into the mRNA for cyclin
• The mRNA was then used to create cyclin in vitro and injected them into Xenopus oocytes
• Exposed oocytes to progesterone
• Known RNA was injected into different oocytes as 25 ng, 5 ng and 2.5 ng.
• Conclusion: there was a pool of inactive pre-maturation-promoting-factor
• Proposal: was present in the cells all the time (although cyclin wasn’t! - went up and down with
cell cycle)
• Therefore, cyclin is a regulatory protein that regulated MPF activity
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Purification of MPF
• Wu and Gerhart
• Purification – separate protein from the rest of cell to usually test activity
• *highly artificial system
• Found that the protein they were purifying would phosphorylate other proteins in a partially
purified sampled
• MPF – Protein Kinase
o MPF – 32 kDa *molecular mass
o Cyclin – 45 kDa – regulatory subunit
• Therefore, MPF phosphorylates many proteins including cyclin
Maturation Promotin Factor Summary
• Entry into mitosis
• MPF is a Kinase – enzyme that adds phosphate to other proteins and causes an conformational
change
• **Fundamental to all cell processes
• Cyclin-dependent kinases (Cdks) – many events within the cell
Using Yeast in Experiments:
• Temperature sensitive mutations
• Some may stop dividing when temperature in increased
• Saccharomyces cerevisiae (CDC28) *commercially available; divides via budding?
• Schizosaccharomyces pombe (cdc2) *divides via fusion?
• Both are useful in looking at the cell cycle
• At a high temperature, cyclin-dependent kinase mutates
• *Can interchange species of proteins (even in mammalian cells); highly conserved
Regulation of the Cell Cycle:
• S. pombe
• Regulation at distinct stages within the cell cycle
• Regulatory event occurs at distinct stages of the cycle
• *Mitotic Cdk activation
• Dependent on cdc2 kinase at 2 points and complete different functions depending on the protein
they are interacting with (cyclins)
• Regulatory points within the pathway: once you commit to starting (critical point) the cycle with
enough resources, the cell will finish cycle
• At point where cell decides to enter mitosis is assessed by regulatory protein
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•
• NOVEMBER 23rd
Checkpoints
• DNA damage/replication – G1, S, G2 *main factor
o If DNA is damaged, cell won’t continue into the next phase
• Can allow occur is the chromosome do not align up correctly at metaphase plate
o Cycle stops if chromosomes are not aligned accurate causes additional or the loss of
some chromosomes
• Sensors – arrest cell cycle progress; cause the cell’s death
o Arrests cycle after recognizing cell damage
o Include:
▪ ATR - G2 Incompletely replicated DNA
• Cell detected single stranded DNA due to replication issue in nucleus
• Proteins binds to region of DNA – ssDNA-protein complex
• ATR binds to complex and becomes active to phosphorylate another
kinase which then phosphorylate cdc25 which activates cdc25
*dephosphorylates C4k (cyclin dependent kinase)
• The inhibitory protein is removed from C4k to
• Cdc25 binds to adaptor protein within cytoplasm
• Cdk kinase being inactivated results in the cell cycle arrestment
▪ ATM – G1 DNA damage double-stranded helix breaks
• MRN recognizes breaks in the double strand and binds to ATM
• ATM activates checkpoint kinase, chk2 which then phosphorylates
transcription factor, p53, so the factor is stabilized (so it is not degraded
quickly)
o Become cancerous if p53 are not working correctly
• *regulating transcription factors regulated gene expression (Stable and
active)
• Affects p21 gene within mRNA which binds to active cdk to cause it to
become inactive and stop the cell cycle
o Kinase are involved in both sensors to bind to transcription factors and cause apoptosis
M Phase: Mitosis and Cytokinesis
• Cell division into two cells with identical genetic content
• Maintains chromosome number; generates new cells
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