Notes past few days

Drs Dohan and Scott provided their own notes in class and say anything they ask will come from there. Here are notes for Kriwacki, though he did not provide all his slides and they are not posted yet. The articles cited in the notes below from Cell Cycle and Genes and Development are freely available, and they pretty well cover the slides we did not get. He handed out the Science article in class and STRONGLY URGED us to read and understand it and the Lodish assigned reading for the exam.
Kriwacki Cell Cycle

Proteins that Regulate Cell Cycle
Read the Kirschner paper about fundamental role of protein degradation in regulation of the cell cycle. Concepts remain central to how aspects of cell cycle are regulated.

Slides are digitally intensive.
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Discoveries were made in yeast due to ease of manipulation.
Cdk inhibition keeps kinases in check.
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Cell cycle in S. Cerevisiae- from text assignment (Lodish 874-890). START is where decisions are made and boundaries crossed to replicate DNA and undergo budding process.
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Screened for genes to complement temperature sensitive mutants. CDC 28 was one of 1st CDK s discovered.
Graphic shows cells harboring a ts mutation. These cells grow at 25 degrees C, but raise the temperature to 37 degrees and they exhibit defects with no budding, even with lots of nutrients. Library of genes in yeast was screened for effects of plasmids in helping yeast to bud.CDC28 was kinase.
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Mechanisms common to all higher organisms. Cancer-cells lose ability to control proliferation.
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Reed late 1980s discovered several cyclin regulatory partners. Similar experiment to earlier ones- factors regulate entry into S phase specifically. Mutation affected interaction of the kinase with its cyclin regulatory partner. Investigators did not know what the regulatory machinery consisted of.
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CDK has regulatory factor bound in order for colonies to form in wild type. In ts cells, the mutant CDC28 protein could only bind to regulatory partner at low temperature. Used library in plasmids to express gene of plasmid at high levels. Able to shift equilibrium to bound and active state.
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CLN3
Approach a little different- screened for plasmid to rescue defect in budding, but used inducible promoter in presence of low glucose. Expressed in absence of glucose. Used FACS analysis of yeast cells to determine whether expressing the gene rescued defect, The cells are labelled with fluorescence tag proportional to the amount of DNA in cell.G1=1 genome. G2= 2 copies. Cell number is on the on y axis. Normal disribution (distribution under normal conditions with normal cells) on left wih 2 peaks. With cyclin vector- not in presence of glucose, increase in peak for actively dividing cells. With glucose-2 traces similar. In C, Yeast defective in G1S phase cyclins with high glucose get stuck in G1. Without- could proceed to G2 with exogenous cyclin, albeit with lower level of replication due to using sick cells.
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Role protein degradation in cell cycle regulation
Inhibitor of CDK is regulated by phosphorylation, which signals protein to be ubiquitinated, causing degradation and relieving inhibition of CDK complex. Mechanism illustrates the way the activity of one set CDK complexes is required to activate another set CDK complexes. One aspect of sequence of events- protein degradation is not easily reversible. Nomenclature is horrible. Clb 5 and 6 are S phase regulation. Inhibitor is Sic1. Cln 1 and 2 phosphorylate Sic1.
For exam, understand concepts, but refer to cyclins and CDKs by general type.
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Diagram puts together results over years. Illustrates way activity of CDK complexes contributes to series of events taking cells into mitosis.
Yeast- only one CDK.Higher organisms- more kinases, more target proteins.
Cln3-CDK phosphorylates a transcription factor that upregulates cln-1 and cln-2 transcription, to pair with CDK to phosphorylate different transcription factors, etc resulting in sequential activation of transcription. Can also trigger degradation to eliminate activity after time needed or relieve repression. CDK has different partners.
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Meshing of gears allowing cells to progress through cell cycle unidirectionally. Once cell enters mitosis, no longer needs active signal. Degrades regulatory subunit, cyclin. Whether to ubiquitinate cyclin depends on G1 CDK complex. Read this in text several times and read review.
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Kirschner’s slides
This is more detail on what we have already covered.
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APC pathway: cells need to have properly set up metaphase plate with appropriate pairing, etc, then go into anaphase. APC targets cyclin B to relieve inhibtion of exit from mitosis. Inactivated CDKs cause cell to sit in G1 until another cell division.
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overview
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Regulation by post-translational modification can be reversed, but regulation by proteolysis cannot.
READ the PAPER for the EXAM!!
Know differences between cyclin types, but not picky names.
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Firing of replication complexes from origins of replication:
In order for replication to occur, must assemble regulatory factors. S phase CDK and DDK phosphorylate proteins involved in pre-replication complex. Phosphorylation signals replication to occur, disassembly of the pre-replication complex, and other factors to come in. RPA binds ssDNA exposed by helicases. Replication complete- origins get loaded w original complex, but in absence of CDK will not become active.
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Mammalian cyclins and CDKs- concepts same, player different.
We have variety of cyclins and CDKs. CDK activates E2F, which phosphorylates retinoblastoma protein, frees E2F, causes transcription cyclins E and A. Follow cyclins through chart.
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last page handout.
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Cell Cycle 4:11, 1491-1494, Nov 2005
CDK1 and 2 may play redundant roles in early stages cell division. This reviews several papers looking for role for CDK1.

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Which kinase dominant in normal cycles? Unknown. Mice wdeveloped normally, so the CDKs 1 and 2 seemed to act together in transitions.
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Alignment of human CDK1 , cdk2, cdk4. Enzymes have distinct roles at different stages. CDK1 is essential for entry into mitosis. Enzymes are really similar. Thr 160 is regulatory Thr phosphorylated for CDKs to be active. Region near it is similar in all these kinases.
PSTAIRE Helix largely conserved.
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Differences in activity come from differences in cyclin regulatory partners. A, B1,D1,E1 alignment. D1 and E are shorter. Much more variability in these sequences. Differences in structure make differences in function.
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Functions:
Genes Dev. 2004: 18: 2699-711.

P107 and 130 Rb phosphorylation, liberates transcription factor to transcribe cyclin E, A, regulatory proteins, resulting in further expression of RB, E2F1, and enzymes involved in nucleic acid synthesis, DNA replication machinery.
Limited targets of kinase liberate a transcription factor controlling a variety of other factors.
E and A, phosphorylate RB at different sites to modify function of protein. P27 is inhibitory.
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cdk activity regulates details in specific examples of cyclins and kinases- Will give copies of slides Monday.
CDK has to be paired with cyclin to have potential to be active.
CAK phosphorylates cyclin/cdk. P21 inhibits activity. Data shows when inhibitor is bound, some complexes are active, so the story is more complicated than we know right now.
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Structural view of CDK2 with key features illustrated.
Kinases have N terminal beta sheet and helical domain at bottom. Active site is interface of these domains in middle in pocket. Regulatory element is PSTAIRE helix- cyclin binds on right and docks on face CDK2 to shove PSTAIRE in and reform active site. Thr phosphorylation is in loop that connects with helical lobe.
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When cyclin A binds, shoves PSTAIRE in. Phospho transfer from ATP occurs.Cyclins interact with substrates.
31These slides may be misaligned.
Phosphorylation introduces negative charge, so basic residues in area are affected. They form pocket and rearrange complex due to phosphorylation. Opens area for substrate binding.
Tues- role of inhibitors and recent findings in field. Read text and paper. Ask questions Tues AM.