Cell Cycle Checkpoints

What is Cell Cycle Proteins

Sequential activation of members of the cyclin-dependent protein kinase (CDK) family promotes the correct timing and ordering of events required for cell growth and cell division . In addition to driving progress through the cell cycle, CDKs are also the downstream targets of checkpoint pathways. These checkpoints act to ensure that critical cell cycle events have been successfully completed before the cell progresses into the next cell cycle stage. They are composed of a surveillance system that detects when a particular cell cycle event has not been correctly executed and a signal transduction pathway whose ultimate target can be a CDK.
Monomeric CDKs are inactive and require both association with a positive regulatory subunit, called a cyclin, and phosphorylation on a conserved threonine residue that lies within the activation loop for full activity. Both the CDK and cyclin families have multiple members, but only CDKs 1, 2, 4 and 6, when bound to their cognate cyclins, appear to have major roles in controlling cell cycle progression. These CDK/cyclin complexes are then additionally controlled by mechanisms that include inhibitory phosphorylation, protein association, subcellular localisation and targeted destruction of regulatory proteins.

How The Cell Cycle Works

The cell cycle consists of four distinct phases: G1 phase, S phase, G2 phase (collectively known as interphase) and M phase. M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's chromosomes are divided between the two daughter cells, and cytokinesis, in which the cell's cytoplasm divides forming distinct cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G0 phase.

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M phase

The relatively brief M phase consists of nuclear division (mitosis) and cytoplasmic division (cytokinesis). In plants and algae, cytokinesis is accompanied by the formation of a new cell wall.

Interphase

After M phase, the daughter cells each begin interphase of a new cycle. Although the various stages of interphase are not usually morphologically distinguishable, each phase of the cell cycle has a distinct set of specialized biochemical processes that prepare the cell for initiation of cell division.

The first phase within interphase, from the end of the previous M phase till the beginning of DNA synthesis is called G1 (G indicating gap or growth). During this phase the biosynthetic activities of the cell, which had been considerably slowed down during M phase, resume at a high rate. This phase is marked by synthesis of various enzymes that are required in S phase, mainly those needed for DNA replication. Duration of G1 is highly variable, even among different cells of the same species.

S phase

The ensuing S phase starts when DNA synthesis commences; when it is complete, all of the chromosomes have been replicated, i.e., each chromosome has two (sister) chromatids. Thus, during this phase, the amount of DNA in the cell has effectively doubled, though the ploidy of the cell remains the same. Rates of RNA transcription and protein synthesis are very low during this phase. An exception to this is histone production, most of which occurs during the S phase.The duration of S phase is relatively constant among cells of the same species.

G2 phase

The cell then enters the G2 phase, which lasts until the cell enters mitosis. Again, significant protein synthesis occurs during this phase, mainly involving the production of microtubules, which are required during the process of mitosis. Inhibition of protein synthesis during G2 phase prevents the cell from undergoing mitosis.

G0 phase
The term "post-mitotic" is sometimes used to refer to both quiescent and senescent cells. Nonproliferative cells in multicellular eukaryotes generally enter the quiescent G0 state from G1 and may remain quiescent for long periods of time, possibly indefinitely (as is often the case for neurons). This is very common for cells that are fully differentiated. Cellular senescence is a state that occurs in response to DNA damage or degradation that would make a cell's progeny nonviable; it is often a biochemical alternative to the self-destruction of such a damaged cell by apoptosis. Some cell types in mature organisms, such as parenchymal cells of the liver and kidney, enter the G0 phase semi-permanently and can only be induced to begin dividing again under very specific circumstances; other types, such as epithelial cells, continue to divide throughout an organism's life.

Cell Cycle Control

The cell cycle, or cell-division cycle, is the series of events that take place in a eukaryotic cell leading to its replication. These events can be divided in two brief periods: interphase—during which the cell grows, accumulating nutrients needed for mitosis and duplicating its DNA—and the mitotic (M) phase, during which the cell splits itself into two distinct cells, often called "daughter cells". The cell-division cycle is a vital process by which a single-celled fertilized egg develops into a mature organism, as well as the process by which hair, skin, blood cells, and some internal organs are renewed.

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Regulation of the cell cycle involves steps crucial to the cell, including detecting and repairing genetic damage, and provision of various checks to prevent uncontrolled cell division. The molecular events that control the cell cycle are ordered and directional; that is, each process occurs in a sequential fashion and it is impossible to "reverse" the cycle.

Role of Cyclins and CDKs

Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases (CDKs), determine a cell's progress through the cell cycle. Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse won the 2001 Nobel Prize in Physiology or Medicine for their discovery of these central molecules. Many of the genes encoding cyclins and CDKs are conserved among all eukaryotes, but in general more complex organisms have more elaborate cell cycle control systems that incorporate more individual components. Many of the relevant genes were first identified by studying yeast, especially Saccharomyces cerevisiae; genetic nomenclature in yeast dubs many of these genes cdc (for "cell division cycle") followed by an identifying number, e.g., cdc25.

Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin. When activated by a bound cyclin, CDKs perform a common biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle. Different cyclin-CDK combinations determine the downstream proteins targeted. CDKs are constitutively expressed in cells whereas cyclins are synthesised at specific stages of the cell cycle, in response to various molecular signals.

General mechanism of cyclin-CDK interaction

Upon receiving a pro-mitotic extracellular signal, G1 cyclin-CDK complexes become active to prepare the cell for S phase, promoting the expression of transcription factors that in turn promote the expression of S cyclins and of enzymes required for DNA replication. The G1 cyclin-CDK complexes also promote the degradation of molecules that function as S phase inhibitors by targeting them for ubiquitination. Once a protein has been ubiquitinated, it is targeted for proteolytic degradation by the proteasome. Active S cyclin-CDK complexes phosphorylate proteins that make up the pre-replication complexes assembled during G1 phase on DNA replication origins. The phosphorylation serves two purposes: to activate each already-assembled pre-replication complex, and to prevent new complexes from forming. This ensures that every portion of the cell's genome will be replicated once and only once. The reason for prevention of gaps in replication is fairly clear, because daughter cells that are missing all or part of crucial genes will die. However, for reasons related to gene copy number effects, possession of extra copies of certain genes would also prove deleterious to the daughter cells.

Mitotic cyclin-CDK complexes, which are synthesized but inactivated during S and G2 phases, promote the initiation of mitosis by stimulating downstream proteins involved in chromosome condensation and mitotic spindle assembly. A critical complex activated during this process is a ubiquitin ligase known as the anaphase-promoting complex (APC), which promotes degradation of structural proteins associated with the chromosomal kinetochore. APC also targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis can proceed.

Specific action of cyclin-CDK complexes

Cyclin D is the first cyclin produced in the cell cycle, in response to extracellular signals (eg. growth factors). Cyclin D binds to existing CDK4, forming the active cyclin D-CDK4 complex. Cyclin D-CDK4 complex in turn phosphorylates the retinoblastoma susceptibility protein (RB). The hyperphosphorylated RB dissociates from the E2F/DP1/RB complex (which was bound to the E2F responsive genes, effectively "blocking" them from transcription), activating E2F. Activation of E2F results in transcription of various genes like cyclin E, cyclin A, DNA polymerase, thymidine kinase, etc. Cyclin E thus produced binds to CDK2, forming the cyclin E-CDK2 complex, which pushes the cell from G1 to S phase (G1/S transition). Cyclin A along with CDK2 forms the cyclin A-CDK2 complex, which initiates the G2/M transition. Cyclin B-CDK1 complex activation causes breakdown of nuclear envelope and initiation of prophase, and subsequently, its deactivation causes the cell to exit mitosis.

Cell cycle inhibitors

Two families of genes, the cip/kip family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) prevent the progression of the cell cycle. Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors.

The cip/kip family includes the genes p21, p27 and p57. They halt cell cycle in G1 phase, by binding to, and inactivating, cyclin-CDK complexes. p21 is activated by p53 (which, in turn, is triggered by DNA damage eg. due to radiation). p27 is activated by Transforming Growth Factor β (TGF β), a growth inhibitor.

The INK4a/ARF family includes p16INK4a, which binds to CDK4 and arrests the cell cycle in G1 phase, and p14arf which prevents p53 degradation. And the amount of chromosomes are able to double at the same rate as in phase 2

The Microtubule Preprophase Band (PPB)

The preprophase band is a microtubule array found in plant cells that are about to undergo cell division and enter the preprophase stage of the plant cell cycle. Besides the phragmosome, it is the first microscopically visible sign that a plant cell is about to enter mitosis. The preprophase band was first observed and described by Jeremy Pickett-Heaps and Donald Northcote at Cambridge University in 1966.

Just before mitosis starts, the preprophase band forms as a dense band of microtubules around the phragmosome and the future division plane just below the plasma membrane. It encircles the nucleus at the equatorial plane of the future mitotic spindle when dividing cells enter the G2 phase of the cell cycle after DNA replication is complete. The preprophase band consists mainly of microtubules and microfilaments (actin) and is generally 2-3 µm wide. When stained with fluorescent markers, it can be seen as two bright spots close to the cell wall on either side of the nucleus.

Plant cells lack centrosomes as microtubule organizing centers. Instead, the microtubules of the mitotic spindle aggregate on the nuclear surface and are reoriented to form the spindle at the end of prophase. It has been suggested that the preprophase band may have functions in properly orienting the mitotic spindle.

The preprophase band disappears as soon as the nuclear envelope breaks down and the mitotic spindle forms, leaving behind an actin-depleted zone. However, its position marks the future fusion sites for the new cell plate with the existing cell wall during telophase. When mitosis is completed, the cell plate and new cell wall form starting from the center along the plane occupied by the phragmosome. The cell plate grows outwards until it fuses with the cell wall of the dividing cell at exactly the spots predicted by the position of the preprophase band.

Cell Cycle & Cell Signaling lecture by Robert A. Weinberg

About Speaker
Robert A. Weinberg '64, PhD '69
Founding Member, MIT Center for Cancer Research

Member, Whitehead Institute Daniel K. Ludwig and American Cancer Society Professor for Cancer Research Department of Biology

Robert A. Weinberg has earned some of the top honors in his field. Most recently, he won the 2006 Landon-AACR Prize for Basic and Translational Cancer Research. He is also a 1997 National Medal of Science awardee.

Weinberg's laboratory discovered the first human oncogene and the first tumor suppressor gene. Today, much of his research focuses on new models of breast cancer development including the stages of tumor invasiveness and metastasis.

He earned his Ph.D. in biology from MIT in 1969, and was one of the Founding Members of the MIT Center for Cancer Research in 1973. He was appointed a professor at MIT in 1982, the same year he joined the Whitehead Institute. Weinberg was named American Cancer Society Research Professor in 1985 and received the Daniel K. Ludwig Professorship for Cancer Research in 1997. He is a member of the National Academy of Sciences and the Institute of Medicine.