CHMI 4237 E Special topics in Biochemistry Cell proliferation 1 – basic machinery Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemistry Laurentian University CHMI 4237 E - Winter 2010

Mitosis Blue: DNA / Green: microtubules CHMI 4237 E - Winter 2010

Mitosis Blue: DNA / Green: microtubules CHMI 4237 E - Winter 2010

Cell cycle Mitosis:  ~1 h http://219.221.200.61/ywwy/zbsw%28E%29/edetail11.htm CHMI 4237 E - Winter 2010

Cell cycle Signals CHMI 4237 E - Winter 2010

So, what are the BIG questions: 1) How does the basic cell cycle machinery work? 2) How does the cell ensure that a given step in the cell cycle is properly completed before moving forward? 3) What are the signals that modulate the cell cycle? CHMI 4237 E - Winter 2010

So, what are the BIG questions: 1) How does the basic cell cycle machinery work? 2) How does the cell ensure that a given step in the cell cycle is properly completed before moving forward? 3) What are the signals that modulate the cell cycle? CHMI 4237 E - Winter 2010

Maturation Promoting Factor (MPF): the engine of the cell cycle  MPF can trigger mitosis when injected Into frog eggs Works even in the presence of protein synthesis inhibitors (e.g. cycloheximide) CHMI 4237 E - Winter 2010

Cyclins: drivers of the cell cycle CHMI 4237 E - Winter 2010

Cyclin levels and MPF activity fluctuate during the cell cycle CHMI 4237 E - Winter 2010

MPF: dimer of a cyclin and a cyclin-dependent kinase (cdk) CHMI 4237 E - Winter 2010

MPF: dimer of a cyclin and a cyclin-dependent kinase (cdk) CHMI 4237 E - Winter 2010

Specific cyclins and cdks pair up to control specific cell cycle events Fission yeast Mammals CHMI 4237 E - Winter 2010

Specific cyclins and cdks pair up to control specific cell cycle events NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | NOVEMBER 2001 | 815 CHMI 4237 E - Winter 2010

Importance of CDKs Nature Reviews | Cancer Volume 9 | March 2009 CHMI 4237 E - Winter 2010 Nature Reviews | Cancer Volume 9 | March 2009

CDK regulation Progress through cell cycle CDK activity Cyclin levels CDK levels While CDK activity varies according to the cell cycle, the level of CDKs is pretty constant; This raises the question: what regulates the activity of the CDKs? CHMI 4237 E - Winter 2010

CDK activation 1 – cyclin binding http://www.new-science-press.com/info/illustration_files/nsp-cellcycle-3-4-3_12.jpg CHMI 4237 E - Winter 2010

Protein kinase structure Upper jaw ATP binding P loop: Gly-rich sequence which binds the phosphates of ATP Lower jaw Substrate binding and phosphotransfer reaction Activation loop (aka T loop): forms a barrier between ATP and substrate inactive kinase Phosphorylation of T loop  change in conformation  kinase activation;

Protein kinase structure http://www.nature.com/nrm/journal/v5/n1/images/nrm1280-i1.jpg http://www.waimr.uwa.edu.au/images/misc/Src-Family-lge.gif http://www.new-science-press.com/info/illustration_files/nsp-protein-3-13-3_25.jpg

CDK activation 1 – cyclin binding http://www.new-science-press.com/info/illustration_files/nsp-cellcycle-3-4-3_12.jpg CHMI 4237 E - Winter 2010

But: cyclin levels do not necessarily correlate with CDK activation…. CHMI 4237 E - Winter 2010

CDK activation 2- Phosphorylation by CDK-activating kinases (CAK) http://www.new-science-press.com/info/illustration_files/ CHMI 4237 E - Winter 2010

CDK activation 2- Phosphorylation by CDK-activating kinases (CAK) http://www.new-science-press.com/info/illustration_files CHMI 4237 E - Winter 2010

CDK regulation 3- Phosphorylation status of Tyr 15  Wee 1 (kinase): phosphorylates Tyr 15, inactivating cdk2  Cdc25 (phosphatase): de-phosphorylates Tyr 15, activating cdk2 CHMI 4237 E - Winter 2010

CDK regulation 4- Inhibition by CDK inhibitory proteins (CKIs) Wild type CHMI 4237 E - Winter 2010

CDK regulation 4- Inhibition by CDK inhibitory proteins (CKIs) p27, p57, p21: obstruct ATP- binding site INK4 family: decrease affinity of CDK 4/6 for D-type cyclins CHMI 4237 E - Winter 2010

CDK regulation 4- Inhibition by CDK inhibitory proteins (CKIs) CHMI 4237 E - Winter 2010

CDK regulation 4- Modulation by CDK inhibitory proteins (CKIs) CHMI 4237 E - Winter 2010

CDK regulation 4- Modulation by CDK inhibitory proteins (CKIs) CHMI 4237 E - Winter 2010

CDK regulation 5- Subcellular localization Cyclin B is kept in the cytosol, away of its targets; Just prior to the onset of mitosis, Cyclin B is phosphorylated; Cyc B phosphorylation masks nuclear export sequences, resulting in its accumulation in the nucleus CHMI 4237 E - Winter 2010

CDK regulation 6- Controlled proteolysis CHMI 4237 E - Winter 2010

Ubiquitin-mediated proteolysis 76 aa / 8.5 kDa peptide Reversible modification In yeast: 20% of all proteins can e ubiquitylated Outcome: PolyUb ( Lys48): Protein degradation MonoUb or PolyUb (Lys 63): protein/protein interactions Often works in tandem with phosphorylation; Enzymatic machinery rivals the one used for phosphorylation: 500 E3 ligases vs 518 kinases 80 DUBs vs 120 phosphatases CHMI 4237 E - Winter 2010

Ubiquitin-mediated proteolysis E1: Activating enzyme Very few in the cell E2: Conjugating enzyme Catalyses the addition of Ub to substrate proteins E3: Ub ligases Responsible for the substrate specificity of the E2 enzyme Lots of them in the cell E1 E2a E2b E2c E3b E3a E3c CHMI 4237 E - Winter 2010

Ubiquitin-mediated proteolysis http://www.nature.com/nrm/journal/v6/n8/images/nrm1701-i1.jpg CHMI 4237 E - Winter 2010

Ubiquitin-mediated proteolysis http://www.wormbook.org/chapters/www_ubiquitinpathways/ubiqfig2.jpg http://www.mshri.on.ca/tyers/about_the_lab.html CHMI 4237 E - Winter 2010

Ubiquitin-mediated proteolysis http://www.scills.ac.uk/images/whatis-1.jpg CHMI 4237 E - Winter 2010

Ubiquitin-mediated proteolysis CHMI 4237 E - Winter 2010

CDK regulation http://www.cella.cn/book/13/images/image027.jpg CHMI 4237 E - Winter 2010

Coordinated regulation of CDKs during the cell cycle http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-19/1940.jpg CHMI 4237 E - Winter 2010

CHMI 4237 E - Winter 2010

But: what do CDKs actually do? 1- G1 phase pRb: Retinoblastoma protein Family of 3 proteins: pRb  105 kDa p107 P130 pRb proteins bind proteins of the E2F family E2F: Family of transcription factors When bound to pRb: suppresses expression of genes required for cell cycle progression After dissociation from pRb: E2F activates the expression of cell cycle- related genes NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 8 | AUGUST 2007 CHMI 4237 E - Winter 2010

pRb In G0: pRb is unphosphorylated pRb binds E2F In early G1: pRb is hypophosphorylated by cyclin D-cdk4/6 In late G1: pRb is hyperphosphorylated by Cyclin E/Cdk2 Allows progression through the cell cycle past the restriction (« R ») point: point of no return when the cell is committed to complete the cell cycle: Before the R point: cells require growth signals to progress in G1 After the R point: growth signals are no longer necessary CHMI 4237 E - Winter 2010

pRb and E2F CHMI 4237 E - Winter 2010

Gene modulation by pRb and E2F Repression Activation Current Opinion in Cell Biology 2007, 19:658–662 p107/p130: pRb CHMI 4237 E - Winter 2010

E2F gene targets Cdk2 Cyclin A Cyclin E CHMI 4237 E - Winter 2010

Passing the R point CHMI 4237 E - Winter 2010

Passing the R point:.  Positive feedback loops ensure the Passing the R point:  Positive feedback loops ensure the irreversibility of the cell cycle CHMI 4237 E - Winter 2010

But: what do CDKs actually do? 2- S phase DNA replication occurs once (and only once) during the cell cycle, during S phase; All the enzymes required for DNA synthesis (nucleotide synthesis, DNA synthesis proper, etc) have been produced prior to entering S phase; At the end of S phase: the two copies of a duplicated chromosomes are physically kept together as sister chromatids via a protein called cohesin. http://www.new-science-press.com/info/illustration_files/nsp-cellcycle-4-0-4_1.jpg CHMI 4237 E - Winter 2010

But: what do CDKs actually do? 2- S phase In eukaryotes, replication forks progress at a rate of ~10-100 bp /sec; The massive size of eukaryotic genome requires the presence of multiple replication initiation sites; But: what prevents a given replication origin to be more than once during the same S phase? http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A2433&rendertype=figure&id=A2448 CHMI 4237 E - Winter 2010

But: what do CDKs actually do? 2- S phase Replication origines are licensed by becoming loaded with MCM proteins; MCM loading requires a proteins called CDC6, itself recruited to replication origins by the protein ORC (Origin Replication Complex); MCM/ORC/CDC6 proteins participate in recruiting the DNA replication machinery However, S-phase cyclin (Cyclin A/Cdk2 in mammals) phosphorylates Cdc6, tagging it for degradation. M-phase cyclins (Cyclin B/Cdk2 in mammals) also phosphorylate CDC6, preventing replication initiation during mitosis; The MCM proteins are displaced by the moving replication fork. CHMI 4237 E - Winter 2010

But: what do CDKs actually do But: what do CDKs actually do? 3- M phase - nuclear membrane dissolution One of the most easily recognized event in early mitosis is the dissolution of the nuclear membrane; This requires the dissolution of the nuclear lamina, a mesh of proteins covering the intra-nuclear side of the nuclear membrane; This is accomplished the phosphorylation of lamins by Cyclin B/cdk2; http://www.bc.biol.ethz.ch/people/groups/ulkutay CHMI 4237 E - Winter 2010

But: what do CDKs actually do But: what do CDKs actually do? 3- M phase - nuclear membrane dissolution http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4&part=A2130&rendertype=figure&id=A2174 CHMI 4237 E - Winter 2010

But: what do CDKs actually do But: what do CDKs actually do? 4- M phase – separation of sister chromatids During prophase: cyclin B/CDK1 phosphorylate and inhibit separase; The protein securin also participates in inhibiting separase This ensure that both sister chromatids stay together during the early part of mitosis; Nature Reviews | Cancer Volume 9 | March 2009 CHMI 4237 E - Winter 2010

But: what do CDKs actually do But: what do CDKs actually do? 4- M phase – separation of sister chromatids Nature Reviews | Cancer Volume 9 | March 2009 Upon reaching anaphase, cyclin B and securin are degraded via the APC/cyclosome (a ubiquitin ligase); This results in separase activation, which cleaves cohesin, allowing the separation of sister chromatids; CHMI 4237 E - Winter 2010

But: what do CDKs actually do? 5- M phase – Exiting mitosis In order to complete mitosis, several events triggered by cyclin/cdks have to be reversed: Disassembly of the mitotic spindle Reformation of the nuclear membrane Decondensation of the chromosomes Also: MCM proteins need to be available for licensing the next DNA replication event; This requires that the activation of cyclins/Cdks be terminated; This is accomplished via the « Anaphase-promoting complex/cyclosome » (APC/C) CHMI 4237 E - Winter 2010

The Anaphase-promoting complex/cyclosome NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006 APC/C is a gigantic ubiquitin ligase (the size of a ribosome); Exists in two separate forms: Bound to Cdc20 (APC/Ccdc20) Bound to Cdh1 (APC/Ccdh1) Recognizes proteins with an amino acid sequence dubbed the Destruction box (D-box) Two main targets of the APC/c: Cyclins Securin PNAS December 22, 1998 vol. 95 no. 26 15374-15381 CHMI 4237 E - Winter 2010

APC/CCdc20 - modulates anaphase and mitotic exit NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006 CHMI 4237 E - Winter 2010

The Anaphase-promoting complex/cyclosome Mitosis: APC/C is phosphorylated by cyclins/cdk1, promoting its association with CDC20; Conversely, cyclin/cdk- mediated phosphorylation of Cdh1 prevent it from associating with APC/C; So: APC/Ccdh1 only arises in late mitosis, after cyclins have been destroyed by APC/Ccdc20 NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006 CHMI 4237 E - Winter 2010

The Anaphase-promoting complex/cyclosome APC/Ccdc20 levels decrease at the end of mitosis because: Low cdk1 activity result in its APC/C dephosphorylation Cdc20 is targeted for degradation by APC/Ccdh1. APC/Ccdh1 ensures that cyclin levels stay low for most of G1, permitting the licensing of the DNA replication origins; NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006 CHMI 4237 E - Winter 2010

Inactivation of APC/Ccdh1 A) Phosphorylation by cyclin A/cdk2 B) EMI-1 expression During G1, E2F triggers the expression of EMI1 (early mitotic inhibitor-1); EMI1 inhibits APC/Ccdh1, permitting the increase of G1 cyclins; C) UBCH10 degradation UBCH10 is an E2 enzyme associated with APC/C UBCH10 is essential for cyclin A degradation. It is also a target of APC/Ccdh1; So, the APC/Ccdh1-mediated degradation of UBCH10 allows the accumulation of cyclin A required in late G1; NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006 CHMI 4237 E - Winter 2010

The Anaphase-promoting complex/cyclosome Genes Dev. 2006 20: 3069-3078 CHMI 4237 E - Winter 2010