Cell cycle

Question 1

What are the three steps of activating a CDK?

Answer 1

Binding of Cyclin to CDK causes protein loop (which obscured the active site) to move away from the active site -> partial activation of CDK.
CDK-activating kinase (CAK) phosphorylates an amino acid near the entrance of the Cdk active site.
Small conformational change leads to full activation of the cyclin-CDK complex.

Question 2

Following DNA damage, which steps have to take place to inhibit progression from G1 phase to S phase?

Answer 2

DNA damage response:
DNA damage initiates a signaling pathway by activating ATM or ATR kinases which associate with the damaged site and phowphorylate target proteins Chk1 and Chk2. -> phosphorylate for example p53 which stimulates transcription of p21 which binds G1/S-CDK and S-CDK complexes and inhibits their activities. (preventing progression from G1 to S phase by G1 arrest)

Question 3

Explain the proteolysis pathway of the M-cyclin

Answer 3

APC/C is activated mid-mitosis by associating with Cdc20 which recognizes amino-acid sequences in the M-cyclin target.
Active APC/C-Cdc20 complex together with E1 and E2 (ubiquitin enzymes) ubiquitylate M-cyclin.
leads to Cdk dephosphorylation, required for completion of M phase.

Question 4

SKP2 (S-phase kinase-associated protein) functions as a receptor protein in the phosphorylation-dependent ubiquitination. During which cell cycle stage(s) are SKP2 levels low and why?

Answer 4

SKP2 targets negative regulators of the cell cycle such as p27, p21 and p57 for degradation, and thereby promotes cell cycle progression during S and G2 phases.
The SCF complex is active from late G1 to early M phase. SKP levels are low durin G1 phase, inhibited by APC/C ligases. Allowing build up of Cdks and progression through the cell cycle.

Question 5

How are cell cycle regulating proteins lowered during different phases of the cell cycle? Include in your answer the different proteins that play a role in this process.

Answer 5

cell cycle regulated by Cdks.
Changes in Cdk activity regulated by cyclins. Cdks are only active when bound to cyclin. The rise and fall of cyclin levels is the primary determinant for Cdk activity. Cdk activity is also controlled by other mechanisms such as; phosphorylation of the kinase active site by Wee1 which inhibits Cdk activity and dephosphorylation by Cdc25 activates.

Question 6

What is the function of the T-loop within cyclin-dependent kinases (CDK) when a CDK gets phosphorylated for full activation? Mention the amino acids which play a role in this process.

Answer 6

In an inactive state, without cyclin bound, the active site is blocked by the T-loop. The binding of cyclin causes the T-loop to move out of the active site, resulting in partial activation of the Cdk. Phosphorylation of Cdk by CAK at a threonine residue in the T-loop further activates the enzyme by changing the shape of the T-loop.

Question 7

What happens between the activation of the RTK and the inactivation of Bad in the PI 3-kinase-Akt pathway?

Answer 7

• Ligand activates RTK
• RTK recruits and activates PI 3-kinase
• produces PI(3,4,5)P3 which derves as docking site for PDK1 and Akt.
• Leads to activation of Akt.
• Akt dissociates and phosphorylates target proteins eg Bad.
• When unphosphorylated Bad holds apoptosis inhibitory protein in inactive states.
• Phosphorylated Bad releases inhibitory proteins, which block apoptosis and promote cell survival.
• Phosphorylated Bad binds ubiquitous cytosolic protein, which keeps Bad out of action.

(Akt also activates mTOR which promotes cell growth)