Molecular Cell Biology (Bio 5068)Cell Cycle I

Ron Bose, MD PhDNovember 13, 2018

G1

M

G2

S

CELL DIVISION CYCLE

MARINE INVERTEBRATES (sea urchin, clam & starfish)

1983: Evans & Hunt.

sea urchin egg (completed meiosis and arrested in G1)

35S-methionine

fertilize

remove samples every 10 min.

examine for protein synthesis in first few cell cycles of sea

urchin egg

Saw 55kDa protein= sea urchin cyclin (periodic protein synthesized throughout the cell

cycle but degraded at the end of mitosis:

DISCOVERY AND NAMING OF CYCLINS

A protein (called “cyclin”) was observed to increase as cells approached mitosis, peak in mitosis and then precipitously disappear as cells exited mitosis.

Two proteins (cyclins A and B) increased as cells approached mitosis, peaked in mitosis and precipitously disappeared as cells exited mitosis.

The cell cycle is primarily regulated by cyclically activated protein kinases

Figure 17-15, 17-16 Molecular Biology of the Cell, 4th Edition

Table 17-1. Molecular Biology of the Cell, 4th Edition

Overview of major cyclins and Cdks of vertebrates and yeast

Malumbres M, Nature Reviews Cancer 2009

Evolution of cell cycle control: from yeast to humans

Cdk activity is regulated by inhibitory phosphorylation and inhibitory proteins

Figure 17-18, 17-19. Molecular Biology of the Cell, 4th Edition

Why is cell cycle progression governed primarily by inhibitory regulation?

Figure 17-20. Molecular Biology of the Cell, 4th Edition

Cell cycle control depends on cyclical proteolysis

UBIQUITIN-MEDIATED PROTEOLYIS

E1 (Ubiquitin activating enzyme)

Binds to Ubiquitin in an ATP-dependent manner

Passes Ubiquitin to E2

E2(Ubiquitin conjugating enzyme or UBC)

At least 12 in yeast

some are specific to a given target

E3 (ubiquitin protein ligase)

Large complex in both clam (cyclosome) and in frog (APC

= anaphase promoting complex). Final transfer of

ubiquitin to substrate can be mediated by E2 alone or E2

acting in concert with E3

Proteosome (26S complex) Structure from archaebacterium solved.

Deshaies RJ and Joazeiro CA.

Annu Rev Biochem. 2009

UBIQUITIN-MEDIATED PROTEOLYSIS

APC =Anaphase Promoting Complex

Required for degradation of substrates at Metaphase to

Anaphase transition ( ie: B-type Cyclins and securin)

Cdc20 : targets cyclin A and B-type Cyclins, securin

Cdh1/ Hct1 : targets Plk1 and B-type cyclins

substrate

Ring finger

UBC(E2)

for

ubiquitination

Apc10

Cdc20Apcx

Apc4

Apc5

Apc7

Cdc27

Apc3

Apc1/

BimE

Cdc23

Apc8

Cdc16

Apc6

Have D or

KEN Box

Cullin= Apc2

Apc11

or

Cdh1/ Hct1

WDrepeat-

containing

proteins

Ubiq

Ubiq

Ubiq

Ubiq

Components:

F Box: adapter

Brings substrate to E3 ligase.

F-box binds to Skp1

Additional protein interaction domains

(PID: WD repeat, leucine-rich repeat) binds to substrate

E2:

UBIQ. Conjugating enzyme (transfers UB to substrate)

Skp1:

Bridges F-box to cullin

Cullin:

Organizes and activates E3 complex

Recruits E2-UBIQ conjugating enzyme

Ring finger protein

Participates in E2 binding and catalysis

SCF Ubiquitin Ligases

O'Connell BC, Harper JW. Curr Opin Cell Biol. 2007

SCF E3 Ubiquitin Ligases

CELL CYCLE REGULATION OF Cdc2

Phosphatase(s)

P

T161

P P P

T14 Y15 T161

Cyclin B

Cdc2

Inhibitory kinase(s)

Activating Kinase(s)

Cyclin B

Cdc2

Cyclin B

Cdc2Cdc2

Cyclin B

Cdc2

INACTIVEINACTIVE INACTIVE

INACTIVEACTIVE

Protein-protein interactions

Reversible phosphorylation

Ubiquitin-mediated proteolyis

Cyclin-dependent Kinase Inhibitor Proteins (CKI’s)

1. CIP/KIP family (p21Cip1, p27Kip1, p57Kip2):a. Binds to Cdk2 and inhibits activity.b. Binds Cdk4/6 and helps assemble complexes with

cyclins.

2. INK4 family (p16, p15, p18, p19). a. Specific for Cdk4 and Cdk6. b. Binds Cdk subunit alone and prevents cyclin binding c. Bind and inhibit Cdk4/6-Cyclin D heterodimers.

G1 Control

Cdk 4 & 6 Cyclin D1, 2, 3

Cdk2Cyclin E

G1

M

G2

S

Cip/Kip proteins(p21, p27, p57)

INK4a proteins(p15,16, 18, 19)

Assembly & Sequestration

Mechanisms controlling G1/S-phase transition

Figure 17-30. Molecular Biology of the Cell, 4th Edition

MITOGENIC

or

HORMONAL

SIGNALS

CYCLIN D Stability and

CYCLIN D-dependent Kinases

(Cdk4/Cdk6)Rb1

E2F

Transcription

factor

CELL CYCLE GENE

XRETINOBLASTOMA PROTEIN

E2F1

HDACs hSWI/SNF

Rb binds to histone deacetylases (HDACs) and ATP-dependent nucleosome

remodeling complexes (hSWI/SNF) thereby promoting nucleosome assembly

and inhibiting transcription. Phosphorylation by cyclin D/Cdk4 and

Cyclin E/Cdk2 displace HDACs and hSWI/SNF from Rb, respectively.

MITOGENIC

SIGNALS

CYCLIN D STABILITY

CYCLIN D-DEPENDENT KINASES

(Cdk4/Cdk6)

RB

E2F

E2F

CYCLIN E

CYCLIN E

CDK2

CYCLIN A &

S-PHASE GENES

S-PhaseS-PHASE GENES

Relief of Rb- mediated

transcriptional repression

S-Phase

G1 Control

MITOGENIC

SIGNALS

CYCLIN D STABILITY

CYCLIN D-DEPENDENT KINASES

(Cdk4/Cdk6)

RB

E2F

E2F

CYCLIN E

CYCLIN E

CDK2

CYCLIN A &

S-PHASE GENES

p27KIP1 p27KIP1-Phosphorylation

Ubiq-Mediated proteolysis

Assembly &

Sequestration

S-PhaseS-PHASE GENES

Relief of Rb- mediated

transcriptional repression

S-Phase

G1 Control

CheckpointsWhat are they?

How were they defined?

How does their derailment contribute to cancer?

CHECKPOINTS

G1G1

MM

G2G2

SS

DNA DAMAGE

DNA DAMAGEUNREPLICATED DNA

IMPROPER SPINDLE ASSEMBLY

STOP!

Checkpoints: intracellular signaling pathways that determine if previous stepsare complete before proceeding onto the next stage (complete DNA synthesis before entering mitosis; spindles must be assembled before exiting metaphase and entering into anaphase) and whether there has been any damage to the DNA.

DNA damage checkpoint: integrity of DNA DNA damage is repaired before entering S, completing S or entering M.

DNA replication checkpoint: replication state of DNA Complete DNA synthesis before mitosis.

Spindle assembly checkpoint: integrity of spindle spindles must be assembled before exiting metaphase into anaphase.

G1

M

G2

S

G1-PHASE CHECKPOINT

S-PHASE CHECKPOINT

G2-PHASE CHECKPOINT

DNA DAMAGE RESPONSE PATHWAY

STOP!

CELLULAR RESPONSES TO CHECKPOINT ACTIVATION

(IR, etoposide, HU, gemcitibine, irinotecan, carboplatin…)

G1 S G2 M

CHECKPOINTS

APOPTOSIS

SENESCENCE

TEMPORARY CELL CYCLE ARREST

& activation of DNA repair pathways

IR EtoposideGemcitabineCytarabine5-Fluorouracil

IrinotecanTopotecan

CisplatinCarboplatin

ATM ATR

CHK2 p53 CHK1

RAD51FAND2FANCE

p2114-3-3s

BAXPUMA

CDC25A

Apoptosis Cell Cycle Arrest DNA Repair

Signaling Cascade

CellularResponse

Chemo- &Radio-therapy

Signal

Fig. 2

Senescence

DNA DAMAGE CHECKPOINTS

Chk1

CyclinE / Cdk2

G1 S G2G1-checkpoint S-phase checkpoint G2 checkpoint

MDEATH

Cdc25A

DNA DSBs ssDNA

ATM

Mdm2p53

p21, 14-3-3s

IR/VP16 replication stress

ATR

Chk2

Cyclin B/Cdk1

Overproduced in certain cancers.

Inactivated in certain cancers.

DNA damage leads to cell cycle arrest in G1

Figure 17-33. Molecular Biology of the Cell, 4th Edition

Figure 17-41. Molecular Biology of the Cell, 4th Edition

Mitogens stimulate cell division

Figure 17-42. Molecular Biology of the Cell, 4th Edition

Excessive stimulation of mitogenic pathways

can lead to cell cycle arrest or cell death

Figure 17-47. Molecular Biology of the Cell, 4th Edition

Extracellular Survival Factors Suppress Apoptosis

Malumbres M, Nature Reviews Cancer 2001

Cell cycle regulators are frequently disrupted in cancer

O’Leary et al., Nature Reviews Clinical Oncology 2016

Overview of CDK inhibitors in clinical

development for cancer therapy

Turner et al, NEJM 2015

NEJM Nov. 3, 2016

Conclusions

1. The cell cycle is a coordinated and tightly organized

process to ensure the successful replication of the cell.

2. Activity of CDK-Cyclins is determined by:1. Synthesis of Cyclins.

2. Reversible phosphorylation/dephosphorylation of

stimulatory and inhibitory sites on CDK.

3. Ubiquitin mediated degradation of Cyclins.

4. CDK inhibitors – INK4 and CIP/KIP families.

3. Checkpoints can halt the cell cycle if all steps have not

been properly completed.

4. Cancers have many alterations in cell cycle proteins

and selective CDK4/6 inhibitors are now used in

cancer treatment.

Question

Which 3 non-mammalian species were

instrumental in our understanding of

the cell cycle?

Prize and Question