Microbiology 22: (Fry) Cdk regulation

Question 1

Structural changes induced by cyclin binding to cdk?

Answer 1

Movement of T-loop out of the catalytic cleft

Rotation of the PSTAIRE helix

Glu-51 and Lys-33 form a salt bridge that enables presentation of the gamma-phosphate of ATP for nucleophilic attack

Question 2

molecules involved in phosphorylation regulation of Cdk1 (Cdc2)? roles?

Answer 2

Cdc2+ (kinase) and Cdc13+ (cyclin) form dimer -> cyclin activates Cdk

Cdc2+ inhibited by Wee1+ (kinase)

‘uninhibited’ by Cdc25+ (phosphatase)

Question 3

Describe regulation process of Cdk1 by phosphorylation

Answer 3

Cdk1(cdc2) is itself a kinase, but can also be phosphorylated to regulate its activity (only in presence of cyclin)

Cyclin binds to Cdk1 -> conformational change
- residues now exposed for phosphorylation

Exposure means molecules can now act

• Wee1 inhibitory kinase can act
• CAK activating kinase can act

Cyclin binding facilitates phosphorylation by CAK

• CAK is always active and in excess
• phosphorylates Thr-161 in activation loop

Cyclin binding also facilitates phosphorylation by Wee1
- phosphorylates T-14 and Y-15 in ATP binding site

Active cdc25 phosphatase removes inhibitory phosphates -> leaves active MPF

Question 4

What is G1/S transition regulated by? Families? How do they work?

Answer 4

Cdk inhibitor proteins (CKIs)

2 families
- p16 (externally stimulated) -> inhibit Cdk4 and Cdk6
Inhibition via blocking of cyclin binding

• p21 (internally stimulated) and p27 (externally stimulated) -> inhibit Cdk2
  Conformational changes dont happen properly

Question 5

How are cyclins degraded?

Answer 5

Destruction motif/sequence near N terminus

recognised by complex of proteins forming enzyme
- Ubiquitin Ligase -> anaphase promoting complex/cyclosome (APC/C)

Covalently attaches ubiquitin to molecules targeted for degradation

Lumen of proteosomes contain peptidase activity -> degradation

Question 6

Consensus site targeted by Cdk1?

Answer 6

S-P-x-K/R or T-P-x-K/R

Question 7

Examples of mitotic Cdk substrates?

Answer 7

Nuclear lamins - disassembly of nuclear envelope
Motor proteins - spindle organisation
Histones - chromatin condensation
Nucleolin - cessation of rRNA synthesis