Post on 31-Mar-2015
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