Wednesday, March 24, 2010

Cell Cycle I: Cdks and cyclins, OH MY

The Cell Cycle
Understanding the cell cycle is critical to comprehending the many other processes that occur.  A myridad of events, such as DNA damage or cell-to-cell signaling, will affect the frequency of a cell’s division, and, as well, the cell cycle will also impact several pathways within the cell.

DNA replication is central to the cell cycle as well, as it must occur once only during the cell’s S phase.  However, before it can do this, the cell must receive growth signals, consider its size and nutrient availability, and determine if there is any DNA damage.  Several checkpoints have been built into the cell cycle prior to S phase to prevent the cell from unnecessarily replicating its DNA, which is a very costly process energetically.   Thus, the cell would not want to waste its resources if it cannot complete replication or if the DNA is damage.  After all, what would be the use of replicating if the cell’s DNA is damaged and progeny cells may not even survive?  Additionally, synthesis of the DNA must also be coordinated with replication machinery that will actually perform the reactions necessary.  Finally, regulators of replication initiation complexes (see notes on DNA replication for information about initiation complexes) must be phosphorylated or synthesized to prepare the DNA for S phase.

Much of the original research into the cell cycle took place in yeast, either of the budding or fission variety (S. cerevisiae or S. pombe, respectively), due to the ease of identifying mutants that were unable to progress through the cell cycle.  Due to yeast’s morphology, one is able to easily decipher which stage of the cell cycle it is in.  For example, in budding yeast, one is able to tell that the cell is in S phase when it has just begun to schmoo and M phase when its chromosomes are segregated.  As mentioned, the original studies in cell cycle considered mutants that were unable to complete the cycle and arrested at different stages.  This was performed by using temperature-sensitive (ts) mutants of the yeast.

Isolating temperature-sensitive cell cycle mutants simplified:
1.      Mutagenize with your favorite mutagen
2.      Screen for temperature sensitive mutants by replica plating
3.      Look for cells that arrest at a uniform stage of the cell cycle
4.      Sort mutants into complementation groups
5.      Transform mutants with plasmid library to identify gene of interest

Microscopically, it is relatively easy to determine when a temperature-sensitive mutant arrested in the cell cycle due to the distinct morphology.  Cells that arrest in the same phase of the cycle look the same, regardless of whether there are nutrients present or not.  For example, if a cell had arrested in metaphase in mitosis, all of the cells would appear to be schmooing with their chromosomes lined up at the metaphase plate when they are placed at the non-permissive temperature (36o).  When grown at the permissive temperature (25o), the cells will continue the cell cycle and will not be synchronized.

The molecular basis of cell cycle control
A great number of molecules are involved in the cell cycle and its regulation.  However, the main complex that is the driving force of the cell cycle is Cdk-cyclin. Cdk, or cyclin-dependent kinase, phosphorylates a number of targets when it becomes activated, and it is only active when it is bound to cyclin, which is a short-lived protein in the cell that is present only during cell division.  In fact, there are two types of cyclins (in yeast): S- and M-cyclin, for synthesis and mitosis cyclins.  Their different binding affinities and abundances during different stages of the cell cycle give Cdk its specificity.  Cdk-cyclin can also be called MPF, for mitosis-promoting factor, when it is the engine driving the G2-to-M transition, or SPF, for S-phase-promoting factor, when used for the G1-to-S transition. 

Cyclin binding isn’t the only requirement for Cdk to become active.  In fact, there are a number of additional steps that must occur for the enzyme to phosphorylate its targets.  Two of the first proteins identified that affects Cdk’s activity were Cdc25 and Wee1.  Wee1, when expressed in excess, led to elongated  yeast cells; while Cdc25 excess led to small cells (cells that passed through mitosis quickly).  Thus, Wee1 acts to inhibit MPF (Cdk-M-cyclin) and Cdc25 activates it.  The exact mechanisms was found to be phosphorylation and dephosphorylation:  Wee1, a kinase, adds an inhibitory phosphate to Cdk on tyrosine 15; Cdc25 phosphatase removes this inhibitory kinase and promotes progression through the cell cycle.  Cdk required both the removal of the inhibitory phosphate and the binding of cyclin to become partially active, but this, too, is not all for a fully active enzyme.  In addition, Cdk must be phosphorylated by a Cdk-activating kinase (CAK), which adds an activating phosphate group to make the enzyme fully active.  This phosphorylation event at threonine 161 on Cdk results in the extension of the T-loop to allow substrate binding and facilitate catalysis.

Due to the importance of Cdk-cyclin for the cell, it has several layers of regulation, and there are many other factors other than phosphorylation, as described above, that regulate it.  When Cdk-cyclin is associated with a CDK inhibitor (CKI) such as p27 (mammalian cells) or Sic1 (yeast), its activity is blocked.  Another CKI is p21, which is involved in DNA damage response.  When DNA damage is detected and p53 is activated, p21 is active and binds Cdk/cyclin to prevent cell cycle progression. CKIs are deactivated by SCF, which bind and ubiquitinated CKI when it is phosphorylated (CKI acts as a phosphordegron – it is targeted for degradation upon phosphorylation).  When SCF is active and ubiquitinates the CKI, Cdk-cyclin can become active.  Sic1 in yeast is involved in binding the S-phase cyclin and Cdc28 (the Cdk), and it is phosphorylated by the Cdk-cyclin present during G1.  In this way, the Cdk-cyclin that is active prior to S-phase cyclin/Cdc28 works to activate the next stage in the cell cycle.

Review: Regulation of Cdk/cyclin
·         Cyclin must bind Cdk for activity
·         Activating phosphate (added by CAK)
·         Inactivating phosphate (added by CKI/Wee1; removed by Cdc25)
·         Inactivation by p21/27
·         p21 is stimulated by p53 during DNA damage

Cyclin D is an important cyclin molecule involved in cell cycle progression that binds Cdk4/6.  When this complex is active, the Rb protein is phosphorylated.  Normally, Rb will be found to E2F, an important transcription factor.  When Rb is phosphorylated, it dissociates from E2F, which then acts as a transcriptional activator to upregulate transcription of Cyclin E / Cdk2 as well as its own transcription and much of the replication machinery.

The cyclincs and Cdks of mammals:
Cyclin D

Cyclin D
G1 – S
Cyclin E
Cyclin A
G2 – M
Cdc2 (Cdk1)
Cyclin B

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