6 cell cycle

G1 Phase

S Phase: DNA replication

G2 Phase

M Phase: Chromosome segregation

1. Cell cycle regulated gene transcription

2. Cell cycle regulated mRNA translation

3. Cell cycle regulated protein proteolysis

4. Cell cycle regulated protein modifications

   • reversible phosphorylation

     -> kinases and phosphatases

• Cyclin dependent kinases are kinases (phosphorylation) which depend on the co-factor cyclin to be activated

• there are different classes of CDKs which differ in the activation (different cyclins) and therefore are active in different phases of the cell cycle

• cyclin dependent kinases are the key drivers of the cell cycle

• Different CDKs are activated by different cyclins, which levels oscillate during the cell cycle

• Therefore different CDK complexes are sequentially activated or inactivated

• the activated CDK phosphorylates many different proteins for example involved in the initial steps of mitosis (for example breakdown of nuclear envelope) or associated with chromosomes (condensation)

• Phosphorylates many hundreds of proteins only in mitosis

• the subunits of the CDKs are present in the cell all the time

• But there is a sequential fluctuation in the presence of the different cyclins

• The cyclins are up regulated by their gene expression

• The down regulation is via protein degradation (prevents re-activation of CDKs)

• the CDK subunits are present in the cell all the time

• The specific cyclin needs to bind to the subunit to partlyactivate the enzyme

• Additionally a phosphorylation in the t-loop by a CDK-activating kinase (CAK) is required

• This enables a switch of activation of the CDKs: first the CDK-cyclin complexes are formed and they are activated by the phosphorylation

• To ensure the “switch-on” activity and that the CDKs are not active before the cell cycle transition happens, they are inhibitory phosphorylated by 2 kinases:

• Wee1= Ser/Thr kinase, Myt1=Tyr kinase

• Wee1 places an inhibitory phosphorylation on the CDK subunit on a Thr residue and Myt1 places a second inhibitory phosphorylation on a Tyr residue

• -> accumulate a lot of CDKs but they are inactive-> switch on activation

• The final activation is Katalysen by the phosphatase cdc25, which de phosphorylates both inhibitory phosphorylations

• down regulation by proteolysis via Ubiquitin and proteasomes

• 2 major Ubiquitin ligases = E3: SCF Complex, APC/C Complex

• Ubiquitin polyasome pathway

• Attachment of ubiquitin to mark the target protein (cyclin) for proteolysis when CDK activity is no longer needed

• Different phases of cell cycle-> different Ubiquitin ligases

• degrading cyclins mainly between G1 and S phase but active throughout the cell cycle

• Substrate recognition by F-box proteins

• Recognition of phosphorylated target proteins

• degradation of S phase cyclins: active in mitosis and G1

• Activating subunits (not F-box): CDC20 and CDH1

• Substrate recognition by sequence motifs: D-box, KEN-Box

• Recognition independent of phosphorylation

1. Cell cycle regulated gene expression (oscillating cyclins)

2. Cell cycle regulated Ubiquitin mediated protein proteolysis (SCF, APC/C)

3. Reversible protein phosphorylation (kinases and phosphatases)

• no only 0.3%

• Differentiated cells (majority) lost ability to under go cell cycle

• Few cells like Epithelial cells or tissue renewal cells can enter the cell cycle (not constantly in the cell cycle) in G1 phase

• To enter the cell cycle these resting cells need growth factors (cancer cells don’t): mitogenic signalling

• Growth factor binds to receptor (dimerisation of receptor)

• Leads to activation of Ras GTPase

• Activates MAP kinase pathway (RAF, MEK, MAPK)

• Phosphorylation of the serum response factor in the nucleus

• SRF = TF: activates a second wave of gene expression-> production of additional TF (Fos, Jun, Myc)

• Fos, Jun, Myc responsible for gene expression of various substrates regulating the cell cycle (cyclin D, cyclin E)

• The unregulated Cyclin D and cyclin E activate specific CDKs which phosphorylate substrates like E2F which allow the entry of the cell cycle by regulating the majority of S phase related genes

• the E2F transcription factors are activated by a cascade, initialised by growth factors/ mitogens (-> Ras-> RAF-> MAPK-> serum response factor-> Fos, jun, Myc-> cyclin D/E -> CDK -> E2F)

• Regulates the replication of cell cycle proteins: cdc6, MCM, RPA

• Activation of CDK (cyclin A)

• Some are activators of transcription and some are repressors

• Pocket proteins mediate the activation of E2F: inhibit the E2F by forming a bond

  -> inhibit the Repressor function or inhibit the activator function

• senescence = permanent exit from cell cycle

• Typical human cell has a division limit = hayfield limit (appr. 50 times)

• Even with high levels of mitogens/ growth factors the cell divides max 50 times

• Due to an accumulation of (oxidative) stress signals and telomere shortening -> DNA damage, oncogene activation

• Counting mechanism is mediated by p16 proteins (=major Tumor suppressor)

• P16 accumulates from cell cycle to cell cycle

• P16 is a CDK inhibitor (increasing number of cell cycles: progressive inhibition of CDK)

  -> no E2F -> no gene expression -> exit of cell cycle

• => preventing DNA damage

• Loss of p16: no chance to control cell cycle clock-> cell cycle would continue without limit-> accumulation of DNA damage -> transformation of cells

• p16 is part of the cell cycle clock and can stop the cell cycle if required (to prevent DNA damage and transformation of cells)

  It inhibits Cdk4 which would normally phosphorylate the pocket proteins to break the bond to the E2Fs

• Cdc4 is a F-box protein which works together with the SCF complex.

  It’s responsible for the degradation of cyclin E.

  The upregulation of Cdc4 would result in a down regulation of cyclin E. This would stop the cell cycle entry

P16, cdc4, pRB

• Major Tumor suppressor, guardian of the genome

• TF which is activated in response to DNA damage -> protective function

• TF p53 has many downstream target genes: p21 protein which is similar to p16. It acts as a CDK 1/ 2 inhibitor

• Stops the cell cycle -> time frame for DNA repair

• Can activate DNA repair genes

• Loss of p53: no response to DNA damage

  Tumor cells continue proliferation even in the presence of DNA damage

• DNA damage on purpose to activate the p53 pathway

  -> stop Cell-cycle and hyper proliferation

• Alternative: CDK inhibitor -> targeting CDK 4

• Problem: Not Tumor specific -> affecting also normal cells

  affecting stem cell renewal, renewal of tissue, epithelial layers…

  -> side effects (for example: loss of hair follicle because they are high proliferating)