Cell Cycle Regulation, Genomic Integrity, and CDKs

Cell Cycle Regulation

• Cell replication (mitosis or meiosis) responds to extracellular signals like growth factors.
• Growth factors bind receptors on the cell surface.
• The binding triggers activation of the cell cycle.
• Cell cycle checkpoints occur at specific points.
• These checkpoints prevent cells from proceeding until certain conditions are satisfied.

Genomic Integrity

• Proper cell cycle control is required to maintain genomic integrity.
• Cell cycle arrest occurs in response to DNA damage allowing adequate time for efficient DNA Repair.
• Improper cell cycling combined wiht faulty DNA repair can lead to a mutated cell.
• Mutates cells lead to abnormal cell division, and cell survival.
• Mutated cells and these abnormalities can result in benign or malignant neoplasms

Cyclin-Dependent Kinases (CDKs)

• Proteins active during each phase regulate events in the cell cycle.
• Master switches that regulate each phase of the cell cycle are cyclin-dependent kinases (CDKs).
• Each phase of the cell cycle is regulated by the activity of a CDK.
• A regulatory subunit called a cyclin regulates CDK activity.
• Combinations of CDKs and cyclin-dependent kinases vary with each phase.

Rules of Cyclin-Dependent Kinase Regulation

• Cyclin-dependent kinases (CDKs) form a complex with a regulatory molecule called a cyclin.
• CDKs must bind to a cyclin to have activity.
• Cyclin/CDK complexes can be regulated to turn off kinase activity, even when the cyclin is still bound.
• The partnering of cyclins and CDKs changes with the phases of the cell cycle.
• Cyclin D and CDK4 or CDK6 present in the G1 (Early) phase
• Cyclin E and CDK2 present in the G1 (Late) phase
• Cyclin A and CDK2 present in the G1/S phase
• Cyclin A and CDK1 present in the S/G2 phase
• Cyclin B and CDK1 present in the M phase

Cyclins and CDKs on a Molecular Level

• Cyclin-dependent kinases are always present in the cell.
• They phosphorylate serine/threonine residues of substrate proteins.
• They are only active when bound to a cyclin.
• Cyclin is a regulatory subunit which is necessary for activity of CDK (structural role, for proper conformation of CDK).
• Cyclin doesn't have kinase activity itself.
• Cyclin is made and degraded as needed during each phase of the cell cycle and is cyclic
• Cyclin confers substrate specificity to the CDK, determining which proteins the CDK can phosphorylate.

Regulation of the Cell Cycle

• Proteins regulated by cyclin/CDKs are needed to carry out the functions specific to that particular phase.
• The CDK portion of the active complex can affect the function of cell-cycle-related proteins in two ways.
• In order to alter the function and/or activity of substrates, CDK phophoryaltes enzymes and other proteins.
• To alter the ability of transcription factors to bind DNA and/or activate transcription, thus altering expression of target genes, CDK phophorylates transcription factors.
• Substrate specificity is also governed by the type of cyclin to which a CDK is bound.
• This ensures that only proteins relevant to a specific cell cycle phase are phosphorylated.

Cyclins Protein Levels

• Cyclin-dependent kinases are present during all cell cycle phases.
• Cyclins are synthesized when needed and degraded when they are not, typically before the next cell cycle phase.
• Specific CDK is only active during a portion of the cell cycle, specifically when complexed with its partner cyclin.
• Cyclin B is only present during M phase.
• Cyclin D is only in G and early S phase.

Cell Checkpoints

• Cyclin-CDKs play a role in three cell cycle checkpoints:
• The Restriction Point (in G1)
• The G2-M checkpoint
• The Spindle Assembly checkpoint (also called the M-phase Checkpoint)
• The Restriction Point checkpoint blocks by default.
• Growth stimulatory signals push the cell through G1 phase.
• The G2/M and Spindle Assembly checkpoints include progression that is always halted until specific conditions are met.

Beginning the Cell Cycle

• Mitogen is extracellular ligand, like a growth factor, which initiates a signaling response ultimately resulting in cell division.
• Mitogens bind to receptors on a cell's surface and initiate phosphorylation cascades, including the MAP kinase cascade.
• MAP kinase increases synthesis and activity of the transcription factor MYC.
• MYC increases expression of the G1-associated cyclin, cyclin D.
• Cyclin D/CDK4/6 is the first active cyclin/CDK complex of the cell cycle.

Dividing or Not

• The cell must cross the restriction point, which is a checkpoint in G1, to proceed through the cell cycle.
• The cell cycle is dependent on mitogen-initiated signaling.
• Progression through G1 begins with the activation of cyclin D-CDK4/6.
• Cyclin D levels are too low to stimulate CDK4/6 activity without a mitogen and the cell enters the quiescent state Go.
• Increased cyclin D expression enables many cell types reenter the cell cycle increasing when exposed to mitogens during a Go state.
• Some cells (nerve cells, and skeletal muscle cells) terminally differentiate in Go and cannot reenter the cell cycle, even when a mitogen is present.

Cyclins at the Restriction Point

• Cyclin D is the only cyclin whose synthesis is dependent upon external signals (mitogens).
• Except for cyclin D, each cyclin is synthesized through the rest of the cell cycle and is initiated by the signaling events of the preceding phase.
• After mitosis, each daughter cell must again be exposed to the same mitogen to progress through G1 to cross the restriction point.
• Cyclin D-CDK4/6 is required but is not sufficient by itself to cross restriction point
• The sequential kinase activities of CyclinD-CDK4/6 (first) and cyclinE/CDK2 (second) allow the cell to pass through the restriction point.
• These proteins work together to maximally phosphorylate (hyperphosphorylate) a protein called Rb.
• The Rb protein derives its name from a retinal cancer called retinoblastoma, which results from the loss of function of the normal Rb protein.
• D-CDK4/6 and E-CDK2 work together to hyperphosphorylate Rb.

Rb and E2F Control The G1-S Transition

• Rb's phosphorylation state determines the cell's transition from G1 to S phase..
• Progression into S-phase relies on the synthesis of proteins.
• Proteins are involved in DNA replication like DNA polymerases and topoisomerases.
• Genes can be transcriptionally activated by E2F, which binds to the promoter regions of S-phase genes.
• E2F remains bound to the promoter region, and binds even when it is inactive.
• Activity of E2F is controlled by the presence or absence of Rb (named for retinoblastoma).
• When Rb is bound to E2F, E2F is inactive

Rb: Regulator of E2F

• When Rb is not phosphorylated, it is bound to E2F to prevent S-phase gene transcription and progression into S phase.
• Rb is phosphorylated in a two-step process by cyclin-CDK complexes:
• In early G1, cyclin D/CDK4/6 phosphorylates Rb.
• In late G1, the cyclin E/CDK2 complex also phosphorylates Rb.
• This two-step phosphorylation of Rb causes Rb to undergo a conformational change that releases Rb from E2F
• E2F is able to stimulate transcription of S-phase genes as it is no longer inhibited by Rb.
• A cell has crossed the Restriction Point when E2F activity enables progression to S phase.

M-Phase Checkpoints

• The M-Phase checkpoints protect DNA from damage.
• DNA is most vulnerable to damage during DNA replication and cell division.
• DNA must replicate correctly, and mitotic chromosomes must be properly distributed to each daughter cell.
• The G2-M and M-phase checkpoints are scheduled halts that protect DNA from damage at these times.
• Both phase transtitions depend on Cyclin B/CDK1.
• In some textbooks CDK1 is referred to as cdc2

The G2-M Phase Checkpoint

• Progression from G2 to M phase requires passage through the G2-M checkpoint.
• Its purpose is to verify that all of the DNA has been replicated correctly.
• Proteins stall the cycle until DNA replication or repair is complete if DNA replication or repair is incomplete.
• The cell signals that it is "ready" to activate B/CDK1.
• CDK1's activation (via cyclin B synthesis and changes in CDK1 phosphorylation) pushes the cell out of G2 and into M phase.

Preparation for the G2-M Checkpoint

• The cell synthesizes cyclin B early in G2, prior to M phase.
• Cyclin B immediately binds to CDK1 in the cell, however cyclin B/CDK1 is not yet activated.
• During G2 phase, CDK1 is phosphorylated; this temporarily inactivates the cyclinB/CDK1 complex.
• The cell cannot cross the G2-M checkpoint until phosphates are removed to activate the complex.

Crossing The G2-m Checkpoint

• As the cell is at G2, CyclinB/CDK1 complexes are stockpiled.
• At this point these complexes are not yet active.
• The cell will generate a ready signal that the DNA synthesis is complete.
• When DNA synthesis is complete this activates a phosphatase called Cdc25.
• Upon activation, Cdc25 dephosphorylates CDK1 removing the inhibitory phosphates.
• This dephosphorylation will activate Cyclin B/CDK1.
• The activated complex will stimulate the events during mitosis.
• The dephosphorylation marks the passage of the cell through the G2-M phase checkpoint.

M-Phase Checkpoint

• The M-Phase or Spindle Assembly Checkpoint is required with cells going through the metaphase step of mitosis.
• During this step the duplicated chromosomes are end-to-end at the metaphase plate.
• A cell is unable to progress any further until duplicated chromosomes are correctly aligned on the spindle.
• Passage through the spindle assembly checkpoint depends on the inactivation of B/CDK1 (via degradation of cyclin B).

Spindle Assembly Checkpoint

• When chromosomes are aligned at the spindle, an "all-clear" signal activates APC, also called, “Anaphase Promoting Comple".
• Following activation, APC, leads to the destruction of cyclin B, the component regulating B/CDK1.
• Cyclin B results in CDK1 molecules that do not have partners, and thus do not have activity, and allows the cell to transition to anaphase.

Response to DNA Damage

• Cyclin-dependent kinase inhibitors (CDKIs) can halt the cell cycle at phase if DNA damage is detected or if conditions for cell division are suboptimal.
• CDKIs bind to the cyclin-CDK complex to inhibit the kinase activity.
• P16 is produced during times of environmental stress and oxygen is (low).
  • p16 ONLY inhibits cyclin D/CDK4 or cyclin D/CDK6 complexes.
• P21 is induced with DNA damage, which is able to inhibit all cyclin/CDK complexes EXCEPT cyclin.
  • D/CDK4 or cyclin D/CDK6.

P21 and Crossing the Restriction Point

• A: YES, p21 can prevent cells from crossing the restriction point.
• The inhibition of CDK activity slows the cell cycle, and provides DNA repair mechanisms adequate time to function.
• True, p21 DOES NOT inhibit cyclin D-4/6
• However, p21 DOES inhibit cyclin E/CDK2.
• Remember that crossing requires the phosphorylation of Rb by both by cyclin D-4/6 and cyclin.
• E/CDK2 With no cyclin E/CDK2 activity, Rb cannot be phosphorylated. Rb is also not active.
• As a result, the cell remains in its G1 Phase.