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Molecular regulation of cell cycle Aleksander L. Sieroń Department of Molecular Biology Lecture presentations are available.

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Presentation on theme: "Molecular regulation of cell cycle Aleksander L. Sieroń Department of Molecular Biology Lecture presentations are available."— Presentation transcript:

1 Molecular regulation of cell cycle Aleksander L. Sieroń Department of Molecular Biology Lecture presentations are available on The course key is „ molecular13”

2 Cyclin levels CDK1 activity DNA replication 2 (4C) DNA content 1 (2C) Segregation of chromosomes Cell divisions 2 Cell size 1 CELL CYCLE Aleksander L. Sieroń

3 Cell cycle:

4 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 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 and morphological changes, from the end of the previous cell division by the end of next one includes a set of biochemical and morphological changes, from the end of the previous cell division by the end of next one

5 Cell cycle phases M-phase (the period of cell division) M-phase (the period of cell division) Interphase (the period between cell divisions): 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).

6 Cell cycle – what happens?

7 Cell cycle – how long it lasts?

8 nuclear mitotic apparatus protein Ribonucleic Acid Export 1 Cell cycle – phases of the M phase

9 Cell cycle: intracellular events.

10 Concentration of cyclins Activity of CDKs DNA replication 2 DNA content 1 Segregation of chromosomes Cell divisions 2 Cell size 1 CELL CYCLE Aleksander L. Sieroń (cyclin-dependent kinases

11 Cell cycle - controling molecules.

12 inhibition activation /p21 p53 or p16/

13 Cell cycle - controling molecules.

14 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

15 Mitosis Mitosis Somatic cells 1 division A result of the division is 2 daughter cells from 1 cell Chromosome numbers Before division 2n Before division 2n After division 2n After division 2n PROPHASEshort Chromosomes composed of 2 chromatides

16 Mitosis METAPHASE Chromosomes are divided into two chromatids, that move to the equatorial plane of the caryokinetic spindle ANAPHASE To cells poles chromatids disperse, as a result of shrinkage of the caryokinatic spindle fibers TELOPHASE Chromatids reach the pole cells Produced are two nuclei with diploid number of chromosomes Cytokinesis occurs Two daughter cells are formed

17 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

18 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.

19 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

20 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

21 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

22 Three waves of cyclins in cell cycle Changes in the level of three major cyclins in the cell cycle They are the molecular basis of the activity change of CDK-cyclin complexes that control the cycle CDK levels are fixed and are present in excess relative to cyclins APC complex degrades cyclin inactivating CDKs

23 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

24 MPF is active in the G2/M transition Active MPF: CDK1 is dephosphorylated by the phosphatase Cdc25 at 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 nucleolus MPF is inactivated at the Metaphase/Anaphase transition, following degradation of cyclin B in anaphase

25 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

26 Activation and inactivation of MPF Activation: Cdc25C = protein phosphtase, dephosphorylation at Tyr15 and Thr14 Inactivation: Wee1 = inactivating kinase, phosphorylation at Tyr15 and Thr14

27 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

28 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.

29 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 complex Cyclin D1-cdk6/cdk6 Rb phosphorylation S phase gene promoters Phosphorylated Rb

30 Aleksander L. Sieroń

31 p53 DNA damage (UV, Ionising radiation, some drugs, etc.) p21 CYKLIN-CDK CYKLIN + CDK * CYKLINA E/CDK2 Rb:E2F ATP ADP ppRb * E2F G1 S A.L. SIEROŃ; 2005/06 Block of MDM4 Block of MDM2 Stabilization of p53 ARREST IN

32 DNA DAMAGE IN CELL NUCLEUS ATM/ATR (ataxia telangiectasia mutated/ATM and Rad3-related) BRCA1 Chk1 – regulatory kinase hCds1/Chk2 ? Cdc25C Kinase Wee1 Cyklin B/Cdk1 G2M ATM, ATR i hCds1/Chk2 are proteins responding to cell damage changing phosphorylation of BRCA gene product Aleksander L. Sieroń ARREST IN

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

34 Cykl komórkowy cdk2, 4 i 6 cyclins A, E i D p53 p21 pRB cdk 1 cykliny A i B Exit to G 0 pRB/RIZ1 Entrance to Apoptosis Entrance to Apoptosis Aleksander L. Sieroń

35 RB1 GENE IN CANCER CELL CYCLE

36 RB PROTEIN (pRB) PHOSPHORYLATION

37 INTERACTION OF pRB AND TRANSCRIPTION REGULATORS Modified from

38 Thank you Aleksander L. Sieroń Department Molecular biology and genetics


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