Molecular regulation of cell cycle

Aleksander L. Sieroń

Department of Molecular Biology

http://biolmolgen.slam.katowice.pl

Lecture presentations are available on

http://www.elearning.sum.edu.plThe course key is

„molecular13”

http://pingu.salk.edu/~forsburg/cclecture.html#reg

Cyclin levels

CDK1 activity

DNA replication

2 (4C)DNAcontent1 (2C)

Segregation of chromosomes

Cell divisions

2Cellsize 1

CELL CYCLECELL CYCLE

Aleksander L. Sieroń

Cell cycle:

Cell cycle:• reproductive cycle of cells consisting of a

sequential phases resulting in cell content doubling (growth, replication of DNA) its division into two new daughter cells

• includes a set of biochemical andmorphological changes, from the end of the previous cell division by the end of next one

Cell cycle phases• M-phase (the period of cell division)• Interphase (the period between cell divisions): G1 - phase of rapid growth and reconstruction of cell

organelles (intense anabolic processes, synthesis of cyclin A, C, D, E, proteins, RNA

S - phase DNA replication (doubling the amount of DNA, weight and volume of cell)

G2 - phase preparatory to enter the cells in mitosis (mitotic spindle protein synthesis, synthesis of cyclin B, the production of the components necessary to play the plasma membrane in telophase of mitosis and cytokinesis).

Cell cycle – what happens?

Cell cycle – how long it lasts?

nuclear mitotic apparatus protein Ribonucleic Acid Export 1

Cell cycle – phases of the M phase

Cell cycle: intracellular events.

http://pingu.salk.edu/~forsburg/cclecture.html#reg

Concentration of cyclins

Activity of CDKs

DNA replication

2DNAcontent1

Segregation of chromosomes

Cell divisions

2Cellsize 1

CELL CYCLECELL CYCLE

Aleksander L. Sieroń

(cyclin-dependent kinases

Cell cycle - controling molecules.

Cell cycle - controling molecules.inhibitionactivation

/p21

p53 or

p16/

Cell cycle - controling molecules.

The protein gene products of the cell cycle are:

• enzymes such as protein kinases that phosphorylate proteins or phosphatases that dephosphorylate proteins

• regulatory proteins that activate or inhibit kinases and phosphatases, or alter the activity of other proteins

Mitosis

Somatic cells

1 divisionA result of the division is 2 daughter cells from 1 cell

Chromosome numbers• Before division 2n• After division 2n

PROPHASEshortChromosomes composed of 2 chromatides

Mitosis

METAPHASEChromosomes are divided into two chromatids, that move to the equatorial plane of the caryokinetic spindle

ANAPHASETo cells poles chromatids disperse, as a result of shrinkage of the caryokinatic spindle fibers

TELOPHASEChromatids reach the pole cells• Produced are two nuclei with diploid number of chromosomes• Cytokinesis occurs• Two daughter cells are formed

Cell cycle regulatory factors

Cyclins – proteins₋ their concentration in the cell changes during cell cycle₋ they form complexes with kinases determining their activity₋ known cyclins: A, B, D1, D2, D3, E

Cyclin dependent kinases (CDKs) – enzymes controlling₋ enzymes conducting protein phoshorylation₋ complex formation with cyclins₋ CDKs activity changes during cell cycle₋ known CDKs: 1, 2, 3, 4, 6, 7

Cell cycle regulation

Is done by running the reaction cascade of protein phosphorylation and dephosphorylation.• Phosphorylation means a transfer of a phosphate group from ATP to the

corresponding amino acid residue of the target protein, catalyzed by a variety of protein kinases.

• Dephosphorylation means removal of a phosphate group from a protein phosphatase-catalyzed.

Protein kinases substrates are different proteins in nucleus and cytoplasm, and most of phosphorylated amino acids in the proteins are tyrosine and threonine.• Protein kinase activity depends on a different set of protein’s control system called

cyclins.• Kinases control the cell cycle protein kinases are called cyclin-dependent (Cdk -

cyclin-dependent called protein kinases).• Kinase activation occurs during critical periods of time (points) of the cell cycle.

Checkpoints (no return) i cell cycle

Checkpoint in late G1 phase controls G1/S transition, called START . It decides to enter the cell to the mitotic cycle.

Checkpoint in late G1 phase controls G2/M transition. It decides to enter the cell to mitosis.

Mitotic spindle checkpoint controls the Metaphase/Anaphase transition.It decides the precise section of all sister chromatids (daughter chromosomes) to the two opposite poles of the cell

CDK inhibitors – a family of proteins p16 and p21 combine with CDK blocking phosphorylation processes responsible for stopping cell cycle checkpoint:

Cell cycle inhibitors₋ P53 – „guardian of the genome" a transcription factor

activator of many genes including p21₋ PRb blocks E2F transcription factor required for the transition from

G1 to S phase₋ mutations of genes coding for p53 and p21 lead to uncontrolled

proliferation or cancer transformation₋ p53 and pRb - the products of tumor suppressor genes₋ Suppressor gene - a gene acting as a brake on the process of cell

proliferation or stabilizes the processes maintaining genetic stability of the cells

Schematic presenting external signals influence on a cell

Many cells require different signals for survival, additional signals to share and still other signals to differentiate.

Most of the cells lacking the respective signal undergoes a kind of suicide, known as programmed cell death, or apoptosis.

A cell undergoing apoptosis

survive

divide

differentiate

die

Three waves of cyclins in cell cycle

Changes in the level of three major cyclins in the cell cycleThey are the molecular basis of the activity change of CDK-cyclin complexes that control the cycleCDK levels are fixed and are present in excess relative to cyclinsAPC complex degrades cyclin inactivating CDKs

MPF Complex = CDK1 + Cyklin B(mitosis promoting factor complex)

• CDK1 is a component of an enzyme dimer

(the enzyme phosphorylates other proteins, structural, regulatory, etc.)

Synonyms CDK1:p34 (a protein with MW 34 kD)Cdc2, because it is encoded by a gene Cdc2 in discovered in yeast

• Cyclin B is a regulatory protein, encoded by the CDC13 gene

MPF is active in the G2/M transitionActive MPF:

CDK1 is dephosphorylated by the phosphatase Cdc25at tyrosine 15 (Tyr15), and threonine 14 (Thr14)

Active MPF phosphorylates structural proteins following:

Histones - the effect is the condensation of chromosomes from prophase to metaphase

Lamina of nuclear lamina - the result is fragmentation of nuclear envelope in prophase

Proteins MAP - the result is the creation of the mitotic spindle

Nucleolin - the effect of dispersion in prophase nucleolusMPF is inactivated at the

Metaphase/Anaphase transition,following degradation of cyclin B in

anaphase

Regulation of cyklina B/Cdk1 complex at subcellular level

Synthesis of cyclin B starts immediately after the replication. Its concentration is increased and the moment when mitosis starts.Its subsequent sharp decline begins an output from mitosis.A sudden decrease in the concentration of cyclin destruction is due to the ubiquitin-dependent system .

K

Activation and inactivation of MPF

Activation: Cdc25C = protein phosphtase, dephosphorylation at Tyr15 and Thr14Inactivation: Wee1 = inactivating kinase, phosphorylation at Tyr15 and Thr14

Cyklina B osiąga maksymalną aktywność na początku profazy. W wyniku aktywności kompleksu cyklina B-Cdk1 dochodzi do kondensacji chromosomów, zaniku błony jądrowej i tworzenia wrzeciona podziałowego.Na początku anafazy kohezyna odpowiedzialna za połączenie się 2 chromatyd jest trawiona przez separazę, co pozwala na rozejście się chromatyd.Przesuwają się one w kierunku biegunów komórki. Separaza podczas cyklu jest związana z sekuryną, która jest ubikwitynowana przez kompleks APC (aktywowany przez białko cdc20). W anafazie dochodzi do rozpadu cyklin i inaktywacji Cdk, co powoduje zanik wrzeciona podziałowego, inicjację cytokinezy i przejście do fazy G1

Mitose progression control

Activation of G1/S–Cdk complexes at starting point through removal of an inhibitor p27

Protein p27 belongs to the family of inhibitors of cyclin-dependent

kinases controls the cell cycle by regulating the activity of CDK-cyclin

complexes participates in the formation of stable complexes of cyclin

D1-CDK4 increases the affinity of the CDK4 to cyclin D1, affects the

level of synthesis of D-type cyclins in the cell and the stability of the cyclin D1

the level of its concentration in a cell is indirectly controlled by a complex of CDK2-cyclin E, which is phosphorylated at position 187, threonine p27 molecule. Phosphorylation is a signal to the proteolytic degradation of p27 protein by protease complex 26S.

Protein p16

functions as a kinase inhibitor, which modulates CDK4/Cdk6 Rb protein phosphorylation, thus affecting cell proliferation

Active Rb protein (in dephosphorylation) is maintained in an inactive state-specific protein that regulates the genes. These proteins are necessary to induce transcription of genes that encode proteins involved in cell proliferation.

Rb phosphorylation by active complex CDK4-cyclin D leads to the inactivation of the Rb protein the release of genes that products lead to cell divisions.

Active complexCyclin D1-cdk6/cdk6

Rb phosphorylation

S phase gene promoters

PhosphorylatedRb

Aleksander L. Sieroń

p53

DNA damage(UV, Ionising radiation, some drugs, etc.)

p21

CYKLIN-CDK

CYKLIN + CDK

*CYKLINA E/CDK2

Rb:E2F

ATP

ADP

ppRb

*E2F

G1 SA.L. SIEROŃ; 2005/06

Block ofMDM4

Block of MDM2Stabilization of p53

ARREST IN

DNA DAMAGE IN CELL NUCLEUS

ATM/ATR(ataxia telangiectasia mutated/ATM and Rad3-related)

BRCA1 Chk1 – regulatory kinasehCds1/Chk2?

Cdc25CKinase

Wee1

Cyklin B/Cdk1

G2 M

ATM, ATR i hCds1/Chk2are proteins responding to cell damage changing phosphorylation of BRCAgene product

Aleksander L. Sieroń

ARREST IN

Crosses of doted lines point to defects in ATM and/or ATR pathways in different cancer cell lines.

Cykl komórkowy

cdk2, 4 i 6cyclins A, E i D

p53

p21

pRB

cdk 1cykliny A i B

Exitto G0

pRB/RIZ1

Entranceto

Apoptosis

Entranceto

Apoptosis

Aleksander L. Sieroń

RB1 GENE IN CANCER CELL CYCLE

RB PROTEIN (pRB) PHOSPHORYLATION

INTERACTION OF pRB AND TRANSCRIPTION REGULATORS

Modified from

Thank you

Aleksander L. Sieroń

Department

Molecular biology

and genetics

http://biolmolgen.slam.katowice.pl