Kriwacki

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.

Kriwacki Cyclins II

Quick run-through of lecture 1:
At end of today will give review session. Nutshell: fundamental concepts of how cell cycle is regulated by CDKs and cyclins. Understand how key experiments were done in yeast and what significance of results were. 1989 cyclin paper- read and be able to describe from experimental and conceptual and significance perspectives. New paper in nature about the identity of 2 proteins phosphorylated by S phase CDK complex in yeast key in regulating DNA replication. Took 10 yrs to find kinase targets. We do not understand all of what the complexes are targeting. Look in January issue of Nature.

Specifically read and understand paper handed out today. Also look at protein degradation as a regulating mechanism. No-going-back type of regulation. Interplay between CDK complex. Understand how protein degradation is used to regulate cell cycle. Text and article by Kirchner hit key concepts. APC is an ubiquitination machine. Be able to describe key elements of a CDK that pertain to cyclin binding and conformational change in activation. Review in1999 JMB pavelitch is about structural results.
Main point of structural details- understand those.

Inhibitors of CDKs:
To activate, bind to cyclin and phosphorylate kinase. Levels of cyclins vary cyclically for control. If this were the only level of regulation, the cell could not prevent multiple rounds of division. Family of proteins called p16 act by binding to CDK 4 and 6 to affect entry into cell cycle at earliest stage. Cyclin D-CDK-4 is phosphorylated and phosphorylates Rb/E2F. Inhibiting CDK 4- prevents Rb phosphorylation and entry into S phase. P15,16,18,19 are CKIs.
p19 binds opposite side from PSTAIRE. It wedges in and locks N terminal lobe into open conformation to prevent binding of cyclin. P19 has 3 repeats of ankyrin motif, which is made up of 2 alpha helices and a beta hairpin.They stack together to form broad surface to form interface with CDK 6. Ankyrin repeat proteins are found in proteins involved in protein-protein interactions.
Lots done on biology of these.
P21 and p27 studied extensively since discovered in mid 90s. p27 inhibits most CDK/Cyclin complexes. IN cells, it promotes activity of CDK4 cyclin D. How? We do not know.
Activity of p27 is regulated by degradation through ubiquitination which is regulated by post-translational modification, specifically phosphorylation of p27.
When cells exit quiescence, they inherit high level of p27. Begins G1, but blocks progress. Needs to be reduced for crossing G1/S boundary. Ubiquitin ligase SCF/ Skp2 increase in mid G1 phase. This ubiquitin ligase targets p27 to reduce its levels so later complexes can be activated.
P27 levels reduced in most human cancers. Loss p27 deregulates G1/S checkpoint.

P27 bound to CDK2/CyclinA structure- binds like a molecular staple, one part to each subunit. C terminal regulatory domain does not interact w cyclin or kinase. Accessible for modification by enzymes. Activity of enzyme is required for phosphorylation p27, but p27 inhibits complex.

Structure function relationship p 11
Conserved domains have conserved structure function relationship. Linker helix domain is also conserved.
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Domain 1, domain LH, domain 2 important.
Single turn at end is important to mechanism. 310 helix contains Tyr residue. Structure is highly dynamic. Flexibility plays a role in its function. 310 helix of p27 inserts into ATP binding procket preventing binding of ATP and substrate. Surface recognition by domain 1 is conserved in other cyclins in cell cycle. Helix has sequence MRAIL in cyclin A,B,D,E. Conserved binding site mediates specificity for p27.
What is conserved about linker is length.
Domain 2 wraps around side of CDK2 and positions 310 helix in active site for inhibition. P27 needs to interact with specific sites in order to inhibit.
Phosphorylation alters function of protein from inhibitor to activator and captive substrate of CDK2.
Domain 1 binds first, then forms basis of specificity.
What about the puzzle? How is p27 eliminated?
Tyrosine kinases can phosphorylate Tyr in 310 helix, causing local change to reactivate kinase to phosphorylate Thr residue and progress into cell cycle. Results tie Tyr kinase signalling into cell cycle.
Y88 sits in ATP binding pocket. Tail has to come close to active site once on a while for T187 to get phosphorylated.

NMR- 2D spectra for free p27 amide nitrogens and protons in peptide. Shifts cluster in the free state because the parts of the molecule average to have similar chemical environments due to the molecule’s flexibility.Right- single conformation with effects of neighbors spread out. Other proteins are not labelled, and are invisible. Most resonances can be labelled.Yellow domain 1, green domain 2, red 310 helix region. Right spectrum is non phosphorylated and reflects inhibitory conformation. Resonance changes are fingerprint of structure when bound to complex. Magnitude of changes shown in bar graph on p. 16
Same experiment with phosphorylated p27 on Y88 prepared in vitro- looked at same pair of spectra and plotted histogram- Pattern of chemical shift changes is same, except for Y88 in 310 helix region. No change free to bound when phosphorylated. Phosphorylation of Tyr causes portion inhibitor to be ejected from active site of complex. How can residue be phosphorylated by Tyr kinase when it is bound in ATP binding pocket? It can’t. Must occasionally swing out and get phosphorylated, then it cant go back in. It is naturally flexible.
Phosphorylation reactivates CDK2. Hypothesized that phosphorylation of the Tyr would promote phosphorylation on Thr. Demonstrated using kinase assay. Phosphorylation of the Thr depends on concentration of complex added to reaction with P23 labelled ATP.
Even if most molecules are bound, can still have small amount free kinase to cause reaction even without phosphorylation Tyr. Graphs illustrate levels phosphorylation. Bimolecular reaction. Tyr phosphorylation ejects helix, tail encounters active site CDK2, Thr phosphorylation (intracomplex reaction with inherent rate depending on how often tail encounters active site)- creates linear concentration dependence.
BcrAbl is Tyr kinase active in some leukemias, constitutively active. Promotes chronic myelogenous leukemia and acute lymphoblastic leukemia. This kinase act causes premature eliminates p27. Phosphorylation at early stage targets p27 for degradation. Lin kinase also does this.
Gleevec acts to target Bcr-Abl to make degradation p27 more normal. The Tyr phosphorylated structure may be a novel drug target.
Be able to answer how this mechanism ties post-translational modification and degradation together. This mechanism ties Tyr kinase signalling into cell cycle control.
Known that short segments of the c-terminal tail are recognized only when Thr is phosphorylated. Parts of CDK2/cyclinA also recognized. There are also several Ks in tail that are probably sites of ubiquitination. What residues ubiquitinated and how it happens mechanistically are not shown yet. Lots of regulation probably goes on that we don’t know about yet.

Review:
Details of CDK2 and features preserved in other CDKs.

Ubiquitination machinery slides page 21- not on exam, but covered elsewhere. SCF complex regulates E and D and p27. APC regulates later stages as covered in Kirschner paper.
Exam format- simple questions to describe some of topics pointed out as he went through today. May give words to include in essay on topic. READ ASSIGNED PAPERS!!