CBIO 3400 Week 16 Study Questions 1 What are Cdks Describe the mechanism controlling the function of Cdks Mechanism of controlling function of CDKs during cell cycle during the cell cycle CDKs Cyclin Dependent Kinases 2 subunits kinase p34 cyclin major role in mitosis S phase entry 1 Kinase binds cyclin 2 Cyclin subunit activates kinase 3 Degrade cyclin to inactivate kinase Components of cyclin degradation pathway 1 Proteasome degrades proteins marked w polyubiquitin 2 APC Anaphase Promoting Complex ubiquitinates M phase cyclin 3 SCF complex ubiquitinates G1 S phase cyclins 4 Proteasome degrades polyubiquitinated proteins 5 CAK CDK Activating Kinase activates phosphate on CDK activating CAK 1 Loop blocks access to catalytic site of CDK 2 CAK phosphorylates T161 in activation loop 3 Loop shifts site opens 6 Phosphatase CDC25 removes inhibitory phosphate on CDK that was originally added by Wee1 kinase 7 CKIs CDK Inhibitors must be degraded to activate G1 S phase CDKs 1 2 Use APC and SCF as examples to explain how the cell cycle control system utilizes proteolysis to control cell cycle progression APC Anaphase Promoting Complex regulates metaphase to anaphase transition works via ubiquitin dependent proteasome system 1 Activation of APC results from activation of MPF Mitosis Promoting Factor 2 APC targets cyclin B for ubiquitination degradation This turns off MPF so cell can exit M phase signals metaphase to anaphase transition 3 APC ubiquitinates geminin which normally blocks DNA replication licensing re replication of DNA Need to get rid of geminin for G1 G2 so licensing can happen again then need to build up levels again for S phase so re licensing can t occur 4 APC degrades securin 5 Separase a protease is now free active It degrades cohesins releasing the sister chromosomes SCF Skip Cullin F Box regulates S phase entry destruction of CKI contributes to S phase entry CKIs are degraded by the proteasome after ubiquitination by SCF complexes 3 How do CKIs regulate the activity of Cdks CKIs CDK Inhibitors regulate CDKs via Wee1 kinase which adds the inhibitory phosphate to the CDK Phosphatase CDC25 must then come along remove that inhibitory phosphate in order for the CDK to be active If there is no phosphatase CDC25 present or it is somehow unable to function then CDK will remain inactive 2 4 Describe 3 major types of checkpoints employed during the cell cycle and how they work Unreplicated DNA Checkpoint stops cycle in G2 inactivation of phosphatase CDC25 which normally removes the inhibitory phosphate added by Wee1 kinase CDK stays inactive process works at G2 for unreplicated DNA 1 ATM ATR kinases activated 2 Chk1 Chk2 kinases activated 3 Phosphorylate CDC25 4 CDC25 PO4 binds 14 3 3 5 CDC25 PO4 is degraded by SCF ubiquitination enzyme 6 MPF cannot be activated since inhibitory phosphate is not removed from CDK DNA Damage Checkpoint stops cycle in G1 S phase or G2 affected by p53 p21 CKI arrests cycle also employs phosphatase CDC25 pathway process 1 DNA damage occurs 2 ATM ATR kinases activated 3 Chk1 Chk2 kinases activated 4 p53 phosphorylated 5 p53 becomes stable active 6 p21 CKI transcribed 7 G1 S phase M phase CDKs turned off 8 Cell cycle arrests in G1 S phase or G2 p53 can also signal to initiate programmed cell death 3 process works at G1 S phase S phase or G2 for damaged DNA 1 ATM ATR kinases activated 2 Chk1 Chk2 kinases activated 3 Phosphorylate CDC25 4 CDC25 PO4 binds 14 3 3 5 CDC25 PO4 is degraded by SCF ubiquitination enzyme 6 MPF cannot be activated since inhibitory phosphate is not removed from CDK Mitotic Spindle Checkpoint unattached kinetochore inhibits APC stops cycle in M phase inactivation of APC MAD2 sequesters CDC20 senses MT attachment to kinetochore tension genetic screens Mitotic Arrest Defficient MAD1 MAD2 block activation of APC C 1 Cannot degrade cohesins 2 MAD2 binds itself MAD1 CDC20 3 At unattached kinetochore catalytically recruit 0 MAD2 eventually sequester all CDC20 in MAD2 CDC20 complex 4 All kinetochores attached MAD1 MAD2 is displaced p31 comet releases CDC20 to activate APC 4
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