Molecular regulation of cell cycle

1. Lecturepresentationsareavailable on http://www.elearning.sum.edu.pl The coursekeyis „molecular13” Molecularregulationof cellcycle Aleksander L. Sieroń Department of MolecularBiology http://biolmolgen.slam.katowice.pl

2. CELL CYCLE Segregation of chromosomes Cell divisions 2 Cell size 1 2 (4C) DNA content 1 (2C) DNA replication CDK1 activity Cyclin levels http://pingu.salk.edu/~forsburg/cclecture.html#reg • Aleksander L. Sieroń

3. Cell cycle:

4. 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

5. 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).

6. Cell cycle – whathappens?

7. Cell cycle – howlongitlasts?

8. Cell cycle – phases of the M phase nuclearmitoticapparatus protein RibonucleicAcid Export 1

9. Cell cycle:intracellularevents.

10. CELL CYCLE Segregation of chromosomes Cell divisions 2 Cell size 1 2 DNA content 1 DNA replication Concentration of cyclins (cyclin-dependent kinases Activity of CDKs http://pingu.salk.edu/~forsburg/cclecture.html#reg • Aleksander L. Sieroń

11. Cell cycle - controling molecules.

12. inhibition p53 or activation Cell cycle - controling molecules. p16/ /p21

13. Cell cycle - controling molecules.

14. The protein geneproducts 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

17. Cell cycle regulatory factors Cyclins– proteins • theirconcentration in the cellchangesduringcellcycle • they form complexes with kinasesdeterminingtheiractivity • knowncyclins: A, B, D1, D2, D3, E Cyclin dependent kinases (CDKs) – enzymes controlling • enzymesconducting protein phoshorylation • complexformation with cyclins • CDKsactivitychangesduringcellcycle • knownCDKs: 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 phasecontrols 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 CDKblocking phosphorylation processesresponsible for stoppingcell cycle checkpoint: Cell cycleinhibitors • P53– „guardian of the genome" a transcription factoractivator of many genes includingp21 • PRbblocks E2F transcription factor required for the transitionfrom G1 to S phase • mutations of genescoding for p53 and p21 lead touncontrolled proliferation or cancertransformation • p53 and pRb - the products of tumor suppressor genes • Suppressor gene - a gene acting as a brake on the process of cell proliferation or stabilizesthe processes maintaining genetic stability of the cells

21. Schematicpresentingexternalsignals influence on a cell survive 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 undergoesa kind of suicide, known as programmed cell death, or apoptosis. divide differentiate A cellundergoingapoptosis die

22. Three waves of cyclins in cellcycle Changes in the level of three major cyclins in the cell cycleThey are the molecularbasis of the activity changeof CDK-cyclin complexes that control the cycleCDK levels are fixed and are present in excess relative to cyclinsAPC complex degrades cyclin inactivating CDKs

23. MPF Complex= CDK1 + Cyklin B(mitosispromotingfactorcomplex) • CDK1 is a component of anenzyme dimer (the enzymephosphorylatesotherproteins, structural, regulatory, etc.)Synonyms CDK1:p34 (a protein with MW 34 kD)Cdc2, becauseitisencoded by a geneCdc2 in discovered in yeast • CyclinB is a regulatory protein, encoded by the CDC13 gene

24. MPF is active in the G2/Mtransition Active MPF:CDK1 is dephosphorylated by the phosphatase Cdc25attyrosine 15 (Tyr15), and threonine 14 (Thr14)Active MPF phosphorylates structural proteins following:Histones - the effect is the condensation of chromosomes from prophase to metaphaseLaminaof nuclear lamina - the result is fragmentation of nuclear envelope in prophaseProteins MAP - the result is the creation of the mitotic spindleNucleolin - the effect of dispersion in prophase nucleolus MPF is inactivated at theMetaphase/Anaphasetransition,followingdegradation of cyclin B in anaphase

25. Regulation of cyklina B/Cdk1 complexatsubcellularlevel K Synthesis of cyclin B starts immediately after the replication. Its concentration is increased and the moment when mitosisstarts.Its subsequent sharp decline beginsanoutput from mitosis.A sudden decrease in the concentration of cyclin destruction is due to the ubiquitin-dependentsystem .

26. Activation and inactivation of MPF Activation:Cdc25C = protein phosphtase, dephosphorylationat Tyr15 and Thr14 Inactivation: Wee1 = inactivatingkinase, phosphorylationat Tyr15 and Thr14

27. Mitoseprogressioncontrol 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

28. Activation of G1/S–Cdkcomplexesatstarting point throughremovalof 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-cyclincomplexes • participates in the formation of stable complexes of cyclinD1-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 cyclinD1 • 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 p16functions as a kinaseinhibitor, whichmodulatesCDK4/Cdk6 Rb protein phosphorylation, thusaffectingcellproliferation Active complex Cyclin D1-cdk6/cdk6 Phosphorylated Rb Rb phosphorylation S phasegenepromoters Active Rb protein (in dephosphorylation) is maintained in an inactive state-specific protein that regulatesthe 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 Rbproteinthe release of genes that products lead to celldivisions.

30. Aleksander L. Sieroń

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

32. DNA DAMAGE IN CELL NUCLEUS ATM/ATR (ataxia telangiectasia mutated/ATM and Rad3-related) ATM, ATR i hCds1/Chk2 areproteinsresponding to celldamagechanging phosphorylation of BRCA geneproduct ? BRCA1 hCds1/Chk2 Chk1 – regulatory kinase Cdc25C Kinase Wee1 Cyklin B/Cdk1 G2 M ARREST IN • Aleksander L. Sieroń

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

34. Cykl komórkowy Entrance to Apoptosis Entrance to Apoptosis Exit to G0 cdk 1 cykliny A i B pRB/RIZ1 p53 p21 pRB cdk2, 4 i 6 cyclinsA, E i D • 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 http://biolmolgen.slam.katowice.pl