Cdc28 Regulate Many Cell Cycle Events. Cdc28 drives entry into S-phase as well as Mitosis. How can one enzyme regulate multiple cell cycle events?

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1 Cdc28 Regulate Many Cell Cycle Events Synchronized cdc28ts cells can t initiate S-phase Cdc28 drives entry into S-phase as well as Mitosis How can one enzyme regulate multiple cell cycle events? 1

2 Cells Use Multiple Cyclins to Regulate Different Cell Cycle Events Cicline M Cicline G1 2

3 Il complesso MPF regola l ingresso mitotico (studi su S. pombe) Il complesso MPF è formato da Cdc28/cicline mitotiche in S. cerevisiae e Cdc2/Cdc13 in S. pombe Il complesso SPF regola l ingresso in S (studi su S. cerevisiae) Il complesso SPF è formato da Cdc28/cicline G1 in S. cerevisiae 3

4 Modello di azione del complesso Cdc28/cicline in S. cerevisiae 4

5 Budding Yeast Cyclin Genes Cyclin Identification Expression Function Cln1 mutant G1 START Cln2 mutant G1 START Cln3 mutant G1 START Clb1 Homology M Mitosis Clb2 Homology M Mitosis Clb3 Homology S Spindle Clb4 Homology S Spindle Clb5 Accidental G1 S-phase entry Clb6 Accidental G1 S-phase entry 5 G1 Cyclins B-type Cyclins

6 Cyclins Nomenclature All contain cyclin box domain Cyclin A Cyclin B s S. pombe cdc13... S. cerevisiae CLB1,2,3,4,5,6 G1 Cyclins S. cerevisiae CLN1,2,3 A - cdc2 binding domain and B s closely related destruction box required for S and M A accumulates earlier, degraded earlier - G1 s more similar to each other -PEST domains (Pro,Glu,SerThr) 6

7 Cyclins can be grouped by expression patterns - CLN1 and CLN2 and CLB5 and CLB6 - CLB3 and CLB4 - CLB 1 and CLB2 CLN1,2,CLB5,6 CLB1,2 [Cyclin] CLB3,4 CLN3 G1 Start Metaphase 7

8 Yeast Cyclin Genes: CLNs and CLBs Quadruple clb1,2 mutant Clb1,2 Spindle assembly anaphase Clb3,4 Activate replication origins Clb5,6 G1 entry Triple cln1,2,3 mutant Dominant CLN Mutations CLN1,2,3 Sense cell size, commit to division

9 Vertebrate Cells, like budding yeast, express multiple cyclins, but they also express several CDK s 9

10 Regulation of G1/S transition (start/restriction point) Identification of mammalian G1 cyclins Human cdna libraries screened for ability to complement CLN3 mutant lead to cloning of three human G1 cyclins: Cyclin C Cyclin D1 Cyclin E Cyclin D2 and Cyclin D3 subsequently identified D-type cyclins: respond to growth factors G0 to G1 transition E-type cyclin: expression is periodic peaks at G1/S transition controls ability of mammalian cells to enter S-phase 10

11 Regulation of G1/S transition (start/restriction point) Identification of mammalian cyclin-dependent kinases (CDKs) Human cdna libraries screened for ability to complement budding yeast CDC28 mutant Three cdna clones identified which could complement CDC28 mutant Mammalian cdk1 acts at G2/M transition cdk2 acts at G1/S transition cdk3 unknown function Cdk4 identified in an anti-cyclin D coimmunoprecipitation experiment Unable to complement CDC28 11

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13 Punti di controllo del ciclo cellulare START Budding yeast PUNTO DI RESTRIZIONE Mammiferi 13

14 Restriction point (START) Point at which cell is irreversibly committed to traversing the cell cycle Mammals: restriction point Yeast: START Cell cycle proceeds without influence from environment (only stopped by damage) Late in G1 14

15 To divide or not to divide: that is the question Yeast cells make decision based on cell size, which is dependent on nutrient availability Mammalian cells make decision based on the presence of protein growth factors called mitogens that stimulate cell growth 15

16 In assenza di fattori di crescita cellule di mammifero si arrestano G0 (cellule quiescenti); quiescenti L aggiunta di mitogeni causa passaggio attraverso il punto restrizione (dopo ore); ore Le cellule in proliferazione entrano S (dopo altre 6-8 ore) ore le in il di in 16

17 Cyclin D is required to pass restriction point Colorazione 16 ore dopo la microiniezione e l aggiunta di BrdU al mezzo. Colorante per DNA R point Anticorpi anti-brdu Anticorpi anti-cicl.d 17

18 Continous expression of Cyclin E shortens G1 18

19 Attività dei complessi Cdk/ciclina dei mammiferi All inizio della fase S le cicline D ed E vengono degradate. I livelli di Cdk4 e 6 crollano repentinamente All inizio della fase S viene sintetizzata la ciclina A che si associa a Cdk2. La distruzione della ciclina A o una sua modificazione inibiscono la sintesi di DNA. Queste proteine non sono necessarie per la progressione nella fase S Il complesso Cdk2/ciclina indispensabile per la progressione nella fase A è S 19

20 Cell-cycle phase-specific Cdk complexes Three classes: G1 Cdk complexes S-phase Cdk complexes Mitotic Cdk complexes (also known as MPF) Cell-cycle phase specificity determined by cyclin type and, in some cells, Cdk type 20

21 Controllo del ciclo cellulare dei mammiferi Ingresso in fase M (complesso MPF) Cdk1/ciclina A Cdk1/ciclina B Ingresso in fase S (complesso SPF) Cdk4,6/ciclina D Cdk2/ciclina E punto di restrizione ingresso in S Progressione in fase S Cdk2/ciclina A 21

22 In addition to forming complexes between specific Cdks and their cyclin binding partners, all complexes require phosphorylation and dephosphorylation to become fully active phosphorylation of thr160/161 is important for the activation of all major Cdk/cyclin complexes catalysed by Cdk activating kinase CAK 22

23 Regolazione della Cdk1 mediante fosforilazione-defosforilazione Siti di inattivazione Sito di attivazione Equilibrio tra fosfatasi (Cdc25) e kinasi (Wee1) 23

24 Another mechanism by which CDKs can regulate Multiple Transitions is by using different CDK inhibitors (CKI s) 24

25 CDK activity is controlled by chemical modification, cyclin synthesis/proteolysis, and: CKIs (Cyclin-dependent kinase Inhibitors) 25

26 CKI s can be specific for particular CDK complexes because their binding can depend upon a specific cyclin CKI Cyclin CDK T-Loop 26

27 Down-regulation of a CKI can, in turn, be regulated by a CDK-mediated phosphorylation because its ubiquitin-dependent proteolysis can be regulated by its phosphorylation state Sic1 Cdc28/Cln1,2 27

28 We can add another limb to our regulation tree Transcriptional Control Ubiquitin-dependent Proteolytic Control Wee1 kinases Cdc25 phosphatases Y15 T160 Kap1 phosphatases Phosphorylation Control Cyclin-dependent Kinase Inhibitors (CKI s) Cdk-Activating Kinases (CAK) Transcriptional Control 28

29 Inibitori delle Cdk Membri diversi di questa famiglia inibiscono Cdk diverse Inibitore cellule animali Cyclin-Cdk complex p15, p16 Cdk4/ciclinaD Cdk6/ciclinaD p21, p27 Cdk4/ciclinaD Cdk6/ciclinaD Cdk2/ciclinaE Cdk2/ciclinaA Inibitore lievito Far1 Cdc2/Cln P40 Cdc2/Clb 29

30 Meccanismi di controllo dell attività dei complessi ciclina/cdk Concentrazione delle cicline -livello di sintesi tramite controllo trascrizionale -proteolisi -localizzazione cellulare Fosforilazione delle CDK Inibitori delle CDK 30

31 Situazioni biologiche in cui la proliferazione è controllata Embriogenesi: proliferazione attiva cicli cellulari brevi rapida successione di fasi di replicazione (S) e mitosi (M) crescita "logaritmica". Differenziamento: graduale attivazione di funzioni specializzate espansione clonale di cellule che si specializzano. contemporaneamente il ciclo di divisione viene modulato. Differenziamento terminale: arresto programmato del ciclo (GO). Reversibilità: cellule differenziate possono ripristinare il programma di divisione in risposta a stimoli (danni meccanici, agenti fisici etc.). Proliferazione neoplastica: innesco della proliferazione in assenza di programma, per mutazione di un gene cellulare o infezione di virus 31

32 Controllo del ciclo cellulare e controllo della proliferazione 2 principali livelli di controllo della proliferazione di cellule eucariotiche: controllo della scelta tra destino proliferativo / entrata in quiescenza (G0) ed eventuale differenziamento della cellula. REGOLATO DALL ESTERN0 controllo delle fasi del ciclo e coordinamento tra i diversi eventi necessari alla divisione cellulare. MECCANISMI DI CONTROLLO INTRINSECI 32

33 Regulation of cell cycle A cell continues through the cell cycle after passing the restriction point (START) START unless it encounters genetic damage If the cell receives a go-ahead signal, it completes the cell cycle and divides otherwise it switches to a nondividing state, the G0 phase. Most human cells are in this phase. Liver cells can be called back to the cell cycle by external cues (growth factors), but highly specialized nerve and muscle cells never divide. Progress though the cell cycle is monitored at four checkpoints 33

34 Cell cycle checkpoints ensure integrity of the genome ensure chromosome segregation does not occur if chromosomes are incorrectly aligned on the mitotic spindle and spindle formation is inhibited during the cell cycle a number of processes take place and they need to be co-ordinated each process involves synthesis, assembly and correct function; all the changes that take place during the cell cycle have to integrated and correctly regulated cell does not enter mitosis until DNA replication is complete and DNA damage is repaired 34

35 Checkpoint machinery Look out for defect and emit a signal SENSORS Transmission of signals throughout the nucleus or cell and amplification TRANSDUCERS Delay cell-cycle progression EFFECTORS 35

36 Four checkpoints 36

37 THE G1/S CHECKPOINT 37

38 GROWTH FACTORS ARE INVOLVED IN PASSING THE G1 CHECKPOINT Cyclin Cyclin 1. Arrival of growth factors from other cells. Cyclin Cyclin 2. Growth factors cause increase in cyclin concentration. Cyclin Cyclin CdK CdK ATP CdK Cyclin 3. Cyclin activates cyclin-dependent kinase. ADP Pi Target protein 4. Kinases activate S phase proteins, leading to cell division. 38

39 Growth factor signalling and transcription Growth factors increase the expression of specific genes: 1. early response genes: rapid increase in mrna and protein levels include transcription factors such as E2F, c-myc, cfos, c-jun 2. delayed response genes: include cell cycle proteins such as Cdks, cyclins expression regulated by early response genes 39

40 Regulated expression of two classes of genes returns G0 mammalian cells to the cell cycle Early-response genes: Transcription factors (E2F) Delayed-response genes: CyclinD, E Cdk2, 4, 6 40

41 Il passagggio attraverso il punto di restrizione dipende dalla attivazione di fattori di trascrizione E2F I fattori E2F attivi stimolano la propria sintesi e quella della Cdk2 e della Ciclina E; Il complesso Cdk2/CyclE porta all aumento di fattori E2F attivi 41

42 E2F is a transcription factor that by itself activates transcription E2F gene expression However, Rb binds to E2F and represses its activation function Rb 42

43 P P P Rb P Rb repression is regulated by early G1 Cyclins-CDKs S-phase gene transcription E2F When Rb repression is inhibited by phosphorylation of Rb by early G1 Cyclin-CDK protein kinases (Cyclin D-CDK4/6), E2F stimulates the expression of genes required for S-phase, including genes encoding DNA polymerases and other proteins required for DNA synthesis, enzymes that synthesize dntps, and genes encoding late G1 Cyclin-CDKs (Cyclin E and CDK2) and the major S-phase Cyclin (Cyclin A). 43

44 The G1/S transition in human cells: Rb (retinoblastoma protein) regulates E2F proteins, which are transcription factors for S phase genes 44

45 Tumor-suppressor genes Genes capable of suppressing the tumorforming potential of transformed cells Tumor suppressor genes normally function to suppress cell growth and division. Tumor-suppressor genes encode : Regulatory proteins eg rb, rb p53 Intra-cellular signaling proteins (nf1neurofibrosarcomas) Cell adhesion proteins (dcc colon carcinomas) 45

46 Tumor-suppressor genes Cells become malignant by losing tumor suppressor gene activity. These act as Mendelian recessive traits. A cell has to be homozygous for a nonfunctional (or missing) tumor suppressor gene for it to have an effect. Analogy Tumor suppressor Lack of suppressor brake pedal no brake pedal 46

47 Gene oncosoppressore Rb Raro tumore dell occhio che colpisce circa un bambino su nati Provocato da due mutazioni consecutive che interessano entrambe le copie del gene Rb 47

48 What is the defect in hereditary retinoblastoma? Compromise this good one, then problems! Rb Normal person: 2 good copies of Rb gene Rb Rb or Hereditary retinoblastoma patient: 1 good copy of Rb gene, 1 defective copy 48

49 Il retinoblastoma erediatrio viene ereditato come carattere autosomico dominante Poiché il gene Rb è localizzato sul cromosoma 13 sia i maschi che le femmine hanno la stessa predisposizione alla malattia 49

50 Perdita dell eterozigosi degli antioncogeni 50

51 Meccanismi genetici alla base del retinoblastoma 51

52 Retinoblastoma and the Two-hit model of carcinogenesis Knudsons two-hit hypothesis: familial cases (high frequency, early onset): retinoblastoma caused by a germline mutation of one Rb allele + an acquired somatic mutation of the remaining allele of the Rb gene = both inactivated sporadic cases (low frequency, late onset): retinoblastom caused by two acquired somatic mutations in both alleles = both inactivated 52

53 Knudson s 2-hit mutation model for retinoblastoma 53

54 RB = tumor suppressor gene RB was the first tumor suppressor to be identified. RB is absent or mutated in at least one-third of all human tumors. Cloning of the retinoblastoma gene mapped to 13q14 (loss of heterozygosity) rb-1 gene cloned Mutated or lost in all cases of retinoblastomas Also found mutated in osteosarcoma and small-cell lung cancer 54

55 Il gene Rb-1 Genetica Frequenza del tumore: 1/ nascite Le cellule tumorali sono prive di entrambi gli alleli Rb-1 funzionali Assetto più frequente: una piccola delezione trasmessa dalla linea germinale, poi una mutazione di senso sull allele omologo dà origine a un clone somatico Associazione di molti casi di tumore con la delezione della banda 13q1.4 Trasmissione mendeliana semplice: predisposizione allo sviluppo di tumori dominante: un cromosoma "difettivo" predispone tutti i portatori a sviluppare tumori; sviluppo di tumori recessivo: il tumore si sviluppa in completa assenza del prodotto, quando entrambi gli alleli sono deleti e/o mutati Studi in sistemi modello trasfezione del gene Rb1 in cellule proliferanti, microiniezione di proteina prb, inibiscono il superamento della transizioneg1/s Topo Rb-/- : letale (difetti nell eritropoiesi e nei tessuti neuronali) 55

56 RB - structure of gene and protein Gene Highly complex: 200 kb with 27 exons and introns from 80bp to 60kb Protein multiple bands Mw= kda nuclear phosphoprotein binds DNA non-specifically Rb contains several functional domains Domains A and B are highly conserved from humans to plants, and they interact with each other along an extended interdomain interface to form the central pocket, which is critical to the tumour suppressor function of Rb 56

57 Caratteristiche strutturali di Rb La proteina Rb può essere suddivisa in quattro domini. Il dominio N è responsabile dell oligomerizzazione della proteina in vitro. I domini A e B, detti A/B pocket, sono responsabili del legame di Rb a vari fattori trascrizionali, come E2F e varie oncoproteine virali, e risultano spesso mutati in vari tumori. Il quarto dominio, detto C pocket è un sito di legame per c-abl. 57

58 RB FAMILY p105 RB; p107; p130 prb binds to the transcriptional activation domain of E2F and blocks activation E2Fs transactivate expression of genes that are important for S phase: dihydrofolate reductase thymidine kinase polymerase histones 58

59 RB s function: a signal transducer connecting the cell cycle clock with the transcriptional machinery Cell cycle clock M G2 Rb G1 S Transcriptional apparatus RB constitutively expressed and relatively stable half-life 12 hours Still induced increase in levels resting G0 cells + mitogenic stimuli = RB level increased 4-6x RB modified by phosphorylation during cell cycle 59

60 G0 prb CycC-Cdk3 Rb a substrate of CycD-CDK CycE-CDK CycA-CDK 60

61 RB is active only within a limited time window during the cell cycle Before the R-point in G1: Rb hypophosphorylated = active repressor of growth (inhibits cell cycle progression) SDS-PAGE: 110 kda After the R-point in G1: Rb hyperphosphorylated = inactive repressor of growth (facilitates cell cycle progression) SDS-PAGE: kda Rb is dephosphorylated at the end of mitosis active repressor M G2 Rb G1 R S Rb P P P Inactive repressor P PP 61 P

62 Gate-keeper model for RB The R-point functions as a door that is kept closed by Rb G1 arrest upon overexpression of Rb Under conditions favourable for proliferation Rb phosphorylated R-door is opened In cells with lost Rb-function the door is left open all the time Such cells will also have lost the ability to respond to growthpromoting/-inhibitory signals Mitogenes (+), TGFβ (-), contact-inhibition (-) Two key elements in this model: upstream signals Rb s phosphorylation status Rb s phosphorylations status downstream effects Rb as signal transducer Cell cycle-clock RB s phosphorylation status RB s phosphorylation status transcription apparatus involved in proliferation 62

63 Gate keeper model M G2 Cdk4/6 Cyclin D G1 S Rb R P P P P P P P E2F released S-phase genes expressed 63

64 E2F liberated by Rb inactivation Rb excert its effects through E2F TFs Rb = inactivated Rb = active repressor R-point E2F = activated! 64

65 RB s phosphorylation status a signal to the transcription apparatus Hypophosphorylated RB binds and inactivates the transcription factor E2F/DP Hyperphosphorylation of RB E2F/DP liberated and free to activate genes necessary for proliferation 65

66 The functional state of the retinoblastoma protein (prb) controls cell proliferation GFs, mitogens... Phosho-pRb becomes inactive cyclin D1 synthesis cyclin D1 P cdk4/6 prb Active cyclin/kinase complex P RNA pol II E2F/DP dimers 5..TTTCCGCG 3 5..TTTCCGCG 3 wt prb represses transcription of cell cycle genes Cell cycle genes off G0 Cell cycle progression G1 66

67 Target genes controlled by activating E2Fs E2F sites common consensus binding site: TTTCGCGC No difference in sequence preference between different E2Fs target genes: E2F controls the transcription of cellular genes that are essential for cell division: cell cycle regulators such as cyclin E, cyclin A, Cdc2, Cdc25A, RB and E2F1, enzymes that are involved in nucleotide biosynthesis such as dihydrofolate reductase, thymidylate synthetase and thymidine kinase 67

68 E2F/DP only active in a window of the cell cycle (late G1 early S) Early G1: active RB E2F/DP turned OFF The R-point: inactivated RB E2F/DP turned ON E2F/DP liberated activation of E2F-dependent promoters Late S: E2F/DP turned OFF again cyclin A/cdk2 phosphorylation of E2F/DP reduced DNA-binding target genes turned off 68

69 HOW ARE S PHASE PROTEINS ACTIVATED? Cyclin CdK Rb E2F Rb ATP Pi ADP 1. In normal cells, Rb protein binds to E2F and shuts down the cell cycle. S Phase proteins Rb Pi E2F 2. If growth factors arrive and activate the cyclin-cdk complex, Rb becomes phosphorylated. E2F E2F mrna DNA 3. E2F is released 4. E2F stimulates the production of S phase proteins. When Rb is mutated, no tie-up of E2F, so constant S phase turn on 69

70 Effetti delle mutazioni del Gene Rb Osteosarcomi Carcinomi: 1) Polmonari 2) Mammari 3) Prostatici 70