Lecture 2 (Regulation of the Cell Cycle from the G1 Phase to the S Phase in Mammalian Cells) Flashcards
Summarise cyclin-dependent kinase regulation in the G1 and S phase.
Mitogen signalling mediates the activation of the MAPK pathway, which, in turn, activates the transcriptional activity of Myc. Myc is a gene regulatory protein which activates Cdk through delayed-response gene expression. The activated G1-Cdk phosphorylates the previously active Rb protein, a process which causes the dissociation of the Rb protein from E2F. E2F is the major transcriptional regulator of S-phase gene and cyclin E/cyclin A, and it’s activation leads to DNA synthesis in the S phase.
Describe the establishment of a restriction point.
The decision of whether the cell should divide or not is made during G1, the period during which cells are responsive to mitogenic growth factors and to TGF-β. The balance between the antagonistic positive and negative signals is interpreted at the restriction point, which serves as a sensor to monitor cyclin-dependent kinase activity. At Cdk levels greater than a particular threshold, cells are committed to completion of the cell division cell cycle.
Cell cultures require growth factors to grow, however, in the absence of growth factors, some cells can still complete the cell cycle. Cells in early- to mid-G1 exit the cell cycle following the removal of growth factors and enter quiescence, or G0, however cells that are past the restriction point can complete cell division. This is referred to as cell autonomous program i.e. cell division is programmed by internal mechanisms, with the restriction point regulated by the Rb protein.
Describe the regulation of pocket proteins at the restriction point.
Rb is a transcriptional regulator, which forms a protein complex with E2F family transcription factors; in this form, E2F is inactive. The E2F transcription factor positively regulates cyclin E and cyclin A expression.
Cyclin D-Cdk4/6 phosphorylates Rb in response to mitogenic stimulation; this hypophosphorylated Rb is less effective at inhibiting E2F, leading to low-level cyclin expression. Rb is further phosphorylated by cyclin E-Cdk2, with this hyperphosphorylation eventually causing Rb to completely dissociate from E2F, thus removing its inhibitory effects upon the transcription factor. Once cyclin E is expressed, Rb is rendered inactive for the remainder of the cell cycle.
This entire process is a regulatory feedback loop, which further enhances E2F transcription.
State the cyclin-Cdk complexes that are specific to each phase of the cell cycle.
Early G1 is regulated by cyclin D-CDK4/6
Late G1 is regulated by cyclin E-CDK2
S phase is regulated by cyclin A-CDK2 or cyclin A-Cdc2 (NB: Cdc2 = CDK1)
Mitosis is regulated by cyclin B-Cdc2
What is the function of CDKI proteins?
CDKI proteins, or cyclin-dependent kinase inhibitor proteins, regulate CDK activity. They are expressed at high levels in G1 and ensure that mitogenic signalling is high to bypass the restriction point.
What is the relationship between kinase activity and DNA replication?
Low kinase activity stimulates DNA replication, whilst high kinase activity actively blocks it.
What are the two families of CDKI proteins?
Ink4 (p15, 16, 18 and 19) inhibit cyclin D-CDK4/6 in early G1
Cip/Kip (p57, 27 and 21) inhibit all other cyclin-CDK complexes.
Describe the inhibitory action of the p27Kip1 CDKI protein on the cyclin A-CDK2 complex.
p27 interacts with two essential sites in the cyclin A-CDK2 complex:
- Interacts with the specificity-determining Cy motif in the cyclin subunit
- Inserts into the catalytic cleft of the CDK subunit
This ensures that the cyclin-CDK complex is inactive by preventing substrate binding and phosphorylation.
Describe the dual roles of CDKIs p21 and p27.
There is a large excess of CDKIs present in the early G1 phase of the cell cycle, with p21 and p27 of the Cip/Kip family inhibiting cyclin E-CDK2 and cyclin A-CDK2. However, in order for cells to exit G1 and begin DNA synthesis, the inhibitory effects of the CDKIs needs to be bypassed. So, in addition to their inhibition of cyclin E-CDK2 and cyclin A-CDK2, p21 and p27 can also bind to cyclin D and CDK4/6. This binding occurs outside of the active site and promotes complex formation of cyclin D-CDK4/6 in its active form. The initiation of cyclin D-CDK4/6 activity aids the exit of the cell from the G1 phase.
Summarise the extra/intracellular factors preventing cell cycle progression.
Extracellular:
-anti-mitogenic signals promote CDKI expression
Intracellular:
- CDKI inhibitor proteins reduce CDK activity, preventing the inactivation of Rb
- Rb acts as a kinase sensor, only once CDK activity passes a threshold is E2F released
Summarise the extra/intracellular factors promoting cell cycle progression.
Extracellular:
-Mitogens (promote cyclin D expression)
Intracellular:
- Sequential cyclin expression ensures that the cell cycle must progress
- Increasing CDK activity maintains Rb inactivation and CDKI inhibition
- After E2F activation and cyclin E/A expression, the cell is committed to completion of cell division (cell autonomous program)
Summarise the cell cycle.
The cell cycle is broadly comprised of two stages: interphase and mitosis. Interphase encompasses the G1 (G = gap), S (S = synthesis) and G2 phases. G1 is where the cell begins to interpret growth signals, and as a result, promoters of cell division are expressed within the cell. The cell then progresses to the S phase, where the genome is precisely duplicated, to leave the cell with a diploid genome of 46 chromosomal pairs. The cell continues to grow and prepare for mitosis during G2, until it reaches the mitotic phase, when chromosomes segregate and the cell undergoes cytokinesis to produce two daughter cells.
