Regulation of the cell cycle by Cyclin-Dependent Kinases

Question 1

CDKs and Checkpoints in the Cell Cycle

Answer 1

• cell division requires growth, exact duplication of genetic information (DNA replication), segregation of genetic material (mitosis) and accurate division of cellular contents (cytokinesis)
• the cell cycle can be separated into four sequential time periods or phases: G1, S, G2, and M
• many sequential steps must be completed during the cell cycle, but in most cells the commitment to proceed to mitosis occurs shortly before the beginning of S phase
• once past this start site or restriction point, most cell types progress through S, G2, and M
• however, there are two other major checkpoints in the cell cycle, times at which the cycle stops until certain conditions have been met: entry into mitosis (G2/M), and the transition from metaphase to anaphase of mitosis (Exit M)

Question 2

Cyclin-Dependent Kinase (cdk)

Answer 2

• a serine/threonine specific protein kinase that has no activity unless combined with a matching regulatory subunit (a cyclin)
• as diagramed the concentration of the cycle, then decreases suddenly (the concentration of the kinase subunit remains constant)
• the gradual increase in cyclin concentration reflects continuous protein synthesis, and the decrease is the result of targeted proteolytic destruction
• the cdk activity increases suddenly, not slowly like the concentration of the cyclin subunit

Question 3

CDK Regulatrion

Answer 3

• addition of one inhibitory phosphate and one activating phosphate
• in reality, inhibition may involve addition of phosphates at more than one site
• there is cdk and then cyclin is added to make an inactive cyclin-CDK complex
• protein kinases add first the inhibitory phosphate and then the activating phosphate
• then there is activating protein phosphatase so there is active cyclin-Cdk complex

Question 4

Mitotic CDK

Answer 4

• activation of the mitotic Cdk involves the addition of phosphates to two different residues of the kinase subunit by two different kinases
• the final step in activation, removal of an inhibitory phosphate by a protein phosphatase, starts a positive feedback loop
• activity of a few molecules of the Cdk as a protein kinase stimulates the phosphatase to make more active Cdk molecule
• this amplification steps leads to a very steep activation curves and makes Cdk activation into almost an all-or no event

Question 5

Inactivation of Cdk

Answer 5

• the cyclin subunit is ubiquitylated by an E3 ubiquitin ligase, targeting the cyclin subunit for proteolytic destruction by the proteasome
• the activating phosphate is removed from the kinase subunit
• the E3 ubiquitin ligase that turns off the mitotic Cdk at the transition from metaphase (aligned chromosomes) to anaphase (chromosome separation) is called APC (Anaphase Promoting Complex)
• it triggers that start of anaphase both by turning off cdk activity, and by promoting chromosome separation by activating the protease Separase
• the actual trigger for the metaphase to anaphase transition is the tension on all kinetochores, which inhibits the activity of a kinase. If one chromosome fails to attach to both poles, the kinase remains active and blocks progression to anaphase. When all kinetochores are under tension, the APC becomes active and triggers separation of chromatids through proteolysis of the cohesion complex

Question 6

Multiple CDKs are needed to regulate cell cycle progression

Answer 6

• each cdk has a unique substrate specificity as well as a unique time during the cell cycle when it is active. Different cyclin/cdk combinations are needed at different points in the cell cycle
• cyclins in G1 (cyclin D + cdk4 and cdk6) are permissive for transition to S phase. Entry into S and progressive through S require additional cyclins (cdk2 + cyclin E and cyclin A) as does the start of mitosis

Question 7

Regulation of CDKs by DNA damage

Answer 7

• progression through the cell cycle is usually inhibited in G1 until signals indicate that cell division is appropriate for the cell and tissue type, and that growth conditions are favorable
• however, the cell cycle may be stopped at any time by signals such as DNA damage, to prevent permanent changes to the genome
• DNA damage will delay progression by increasing synthesis and phosphorylation of p53, which both stabilize the protein against proteolytic degradation, and activate it
• once the damage has been required, the cell cycle continues
• under come circumstances, activation of the p53 pathway will lead to cell death (apoptosis) rather than cell cycle arrest

Question 8

Breast cancer and DNA damage repair

Answer 8

• the BRCA1 and BRCA2 proteins are important in DNA double-strand break repair
• if either one is completely absent, then the cell is unable to repair double strand breaks efficiently and therefore mutation rate increase
• most cells with DNA damage do not progress through the cell cycle, but prolonged checkpoint activation usually leads to cell death by apoptosis
• the PARP pathway is important for single-strand break repair
• by inhibiting the PARP pathway in BRCA-mutant cancer cells, and inducing DNA damage with Cisplatin, the rate of apoptosis can be increased

Question 9

External controls of the cell cycle

Answer 9

• external signals work through modulation of cell growth (growth factors) needed to pass the start checkpoint in G1, and through direct activation of Start (mitogens)
• growth factors increase the rate of protein synthesis and related metabolic processes and reduce rates of protein degradation, allowing cells to reach critical growth status for entry into the cell cycle
• factors considered to act as mitogens directly stimulate progression from Go into the cell cycle. These include many ligands of receptor tyrosine kinases that active the MAPK pathway
• pathway activation in turn activates transcription of genes that encode G1 CDK subunits. Activity of the G1 CDK inactivates the retinoblastoma protein (Rb) and triggers synthesis of genes needed to progress into S phase