17.2 Regulators of Cell Cycle Progression

Identification and Discovery of the Prototype Regulators of Cell Cycle Progression (MPF)

The study of cell cycle regulation began with the identification of Maturation Promoting Factor (MPF), which serves as the prototype for regulators of cell cycle progression. Early experiments utilized Xenopus oocytes, which are naturally arrested in the $G_2$ phase of the cell cycle. Under normal physiological conditions, entry into the $M$ phase of meiosis in these oocytes is triggered by the hormone progesterone. However, researchers discovered that microinjecting cytoplasm from a donor oocyte already in $M$ phase into a $G_2$-arrested recipient oocyte would trigger entry into mitosis without the need for hormonal stimulation. This demonstrated the existence of a specific cytoplasmic factor, named MPF, that is sufficient to induce the $G_2 \rightarrow M$ transition.

MPF is a heterodimer composed of two distinct subunits: $Cdk1$, a cyclin-dependent kinase that provides catalytic activity, and cyclin $B$, a regulatory subunit. The activity of MPF is observed to cycle in tandem with the accumulation and degradation of cyclin $B$. This complex initiates mitosis by phosphorylating key substrates that facilitate critical cellular changes, including nuclear envelope breakdown, the formation of the mitotic spindle, and chromosome condensation. The discovery of MPF established the fundamental model for cell cycle control across eukaryotes, illustrating that progression is governed by specific kinase–cyclin complexes.

Evolutionary Conservation and the Discovery of Cdk1 and Cyclins

The identification of $Cdk1$ (Cell Division cycle Kinase $1$) occurred through genetic studies of the yeast Saccharomyces cerevisiae. Specifically, temperature-sensitive $cdc28$ mutants were utilized. At permissive temperatures, these mutants replicate normally; however, when shifted to nonpermissive temperatures, the cell cycle is blocked at a specific checkpoint known as START. The identification of $Cdk1$ homologs in all eukaryotes proved that the mechanisms of cell cycle control are evolutionarily conserved.

Parallel to the work in yeast, cyclins were discovered in sea urchin embryos. These proteins exhibit a unique cyclic pattern: they accumulate steadily throughout interphase and undergo rapid, complete degradation toward the end of mitosis. This periodic synthesis and destruction correspond precisely with the activation and inactivation of the $Cdk1$ kinase. In the universal engine of mitotic entry, $Cdk1$ serves as the catalytic component, while cyclin $B$ controls the timing of activation.

Structural Activation and Regulation by Phosphorylation and Proteolysis

The Maturation Promoting Factor is structurally a dimer where cyclin $B$ binding is absolutely required for the catalytic activity of the $Cdk1$ kinase. The regulation of this complex involves a sophisticated balance of phosphorylation and dephosphorylation events. During the $G_2$ phase, $Cdk1$ binds to cyclin $B$ to form an initially inactive complex.

The activation process involves multiple kinases and phosphatases:

1. CAK (Cdk-activating kinase): Phosphorylates $Cdk1$ at threonine-$161$ ($Thr161$), an activating site required for kinase activity.

2. Wee1 kinase: Phosphorylates $Cdk1$ at tyrosine-$15$ ($Tyr15$) and, in vertebrate cells, at threonine-$14$ ($Thr14$). These are inhibitory sites that keep MPF inactive despite cyclin binding, ensuring the cell does not enter mitosis prematurely.

3. Cdc25 phosphatase: At the transition from $G_2$ to $M$, this phosphatase removes the inhibitory phosphates from $Tyr15$ and $Thr14$, thereby fully activating MPF and triggering the onset of mitosis.

The inactivation of MPF at the end of mitosis is just as critical as its activation. This is achieved through the ubiquitination of cyclin $B$ by the Anaphase-Promoting Complex/Cyclosome (APC/C). Once ubiquitinated, cyclin $B$ is targeted for proteasomal degradation. The loss of the cyclin subunit inactivates $Cdk1$, leading to mitotic exit and the resetting of the cell cycle for the next round. Following cyclin degradation, $Cdk1$ is dephosphorylated to return to its basal state.

Families of Cyclins, Cdks, and Cdk Inhibitors (CKIs)

In animal cells, the cell cycle is driven by a succession of distinct cyclin–$Cdk$ pairs, each governing a specific phase. This sequential activation ensures that the cell cycle moves in a unidirectional and irreversible manner. The progression follows a specific hierarchy:

1. $G_1$ Phase: $Cdk4$ and $Cdk6$ pair with $D$-type cyclins (Cyclin $D1$, $D2$, $D3$). These complexes regulate cell growth and the passage through the restriction point ($R$ point).

2. $G_1 \rightarrow S$ Transition: $Cdk2$ pairs with Cyclin $E$ to initiate DNA replication.

3. $S$ Phase and $G_2$ Progression: $Cdk2$ pairs with Cyclin $A$ to maintain DNA synthesis and prepare the cell for the mitotic transition.

4. $G_2 \rightarrow M$ Transition and Mitosis: $Cdk1$ pairs with Cyclin $A$ and Cyclin $B$ to drive chromosome segregation and mitotic events.

A useful mnemonic for remembering the cyclin order is "DEAB": $D$ for Decision (the restriction point), $E$ for Entry (into $S$ phase), $A$ for Advance (through $S$ and $G_2$), and $B$ for Breakdown (mitotic breakdown of the nuclear envelope).

Negative regulation is provided by Cdk inhibitory proteins (CKIs), which block activity when internal or external conditions are unfavorable. These are categorized into two families:

• Ink4 family ($p15$, $p16$, $p18$, $p19$): These specifically inhibit $Cdk4$ and $Cdk6$ during the $G_1$ phase.

• Cip/Kip family ($p21$, $p27$, $p57$): These inhibit $Cdk2$/Cyclin $E$ during the $G_1 \rightarrow S$ transition and $Cdk2$/Cyclin $A$ during $S$ and $G_2$ phases.

Growth Factor Signaling and the Regulation of the G1 Restriction Point

Progression through the $G_1$ phase is largely determined by external growth factors which regulate the activity of $Cdk4$ and $Cdk6$ by inducing the synthesis of $D$-type cyclins. This occurs via the Ras/Raf/MEK/ERK signaling pathway. Specifically, mitogens activate the Ras/MAPK pathway, leading to the activation of the extracellular signal-regulated kinase (ERK). Activted ERK then stimulates the $Elk-1$/$SRF$ (Serum Response Factor) transcription factor complex. This complex induces the expression of $AP-1$ (consisting of $Fos$ and $Jun$) transcription factors, which directly bind to and activate the promoter for $D$-type cyclin genes.

Once synthesized, Cyclin $D$ binds to and activates $Cdk4$ and $Cdk6$. These active complexes then phosphorylate the Retinoblastoma protein ($Rb$), which acts as a tumor suppressor and a molecular "brake" on the cell cycle. In its underphosphorylated state, $Rb$ binds to and sequesters $E2F$ transcription factors, blocking the expression of genes required for DNA synthesis. Phosphorylation by $Cdk4,6$/Cyclin $D$ causes $Rb$ to dissociate from $E2F$. Once released, $E2F$ activates the transcription of its target genes, including Cyclin $E$, $Cdk2$, and various proteins necessary for DNA replication.

Positive Feedback Loops and Commitment to S Phase

The transition through the restriction point is reinforced by several positive feedback loops that make the process irreversible:

1. $Rb$ Hyperphosphorylation: The initial release of $E2F$ leads to the production of Cyclin $E$ and $Cdk2$. The resulting $Cdk2$/Cyclin $E$ complexes further phosphorylate $Rb$. This amplifies $E2F$ activity, ensuring that even if growth factor signaling ceases, the cell remains committed to DNA replication.

2. Relief of $p27$ Inhibition: In early $G_1$, $Cdk2$/Cyclin $E$ is inhibited by the Cdk inhibitor $p27$. Growth factor signaling inhibits the synthesis of $p27$. Furthermore, as $Cdk2$ becomes active, it phosphorylates $p27$, targeting it for proteasomal degradation. This creates a loop where active $Cdk2$ leads to more active $Cdk2$.

3. APC/C Inhibition: $Cdk2$/Cyclin $E$ phosphorylates and inhibits the APC/C ubiquitin ligase. Since APC/C is responsible for degrading cyclins, its inhibition prevents the degradation of Cyclin $E$, stabilizing the complex and ensuring entry into $S$ phase.

Clinical implications are significant, as the loss of $Rb$ or the hyperactivation of Cyclin $D$/$Cdk4,6$ complexes removes the essential $G_1$ control mechanism, a common hallmark of human cancer development.

Initiation of DNA Replication and DNA Damage Checkpoints

$S$ phase regulation ensures that the genome is copied exactly once per cell cycle. Rising levels of $Cdk2$/Cyclin $E$ trigger the activation of the CMG helicase at replication origins, unwinding the DNA and recruiting DNA polymerases to begin synthesis. Concurrently, high $Cdk$ activity blocks the assembly of new replication complexes at origins that have already fired, preventing re-replication.

Cells employ DNA damage checkpoints to halt the cycle and allow for repair. These checkpoints rely on sensor proteins that activate master kinases:

• ATR: Responds primarily to single-stranded or unreplicated DNA; it activates the signal transducer kinase Chk1.

• ATM: Responds primarily to double-strand breaks; it activates the signal transducer kinase Chk2.

Both Chk1 and Chk2 phosphorylate and inhibit Cdc25 phosphatases. Because $Cdc25$ is required to activate both $Cdk2$ and $Cdk1$, its inhibition results in cell cycle arrest at the $G_1$, $S$, or $G_2$ checkpoints.

The p53 Pathway and Cell Cycle Arrest

The $p53$ protein serves as a critical transcriptional regulator that enforces arrest following DNA damage. Under normal conditions (the basal state), $p53$ levels are kept very low because it is constantly ubiquitinated by the E3 ligase MDM2 and subsequently degraded by the proteasome.

When DNA damage occurs, ATM and Chk2 phosphorylate $p53$. This phosphorylation event prevents MDM2 from binding, allowing $p53$ to accumulate rapidly in the nucleus. Once stabilized, $p53$ acts as a transcription factor to induce the expression of the Cdk inhibitor $p21$. The $p21$ protein then binds to and inhibits $Cdk2$/Cyclin $E$ and $Cdk2$/Cyclin $A$ complexes, halting the cell cycle in the $G_1$ or $S$ phase. This pause provides the cell with the necessary time for DNA repair; however, if the damage is persistent and cannot be fixed, the signaling pathway may ultimately trigger apoptosis (programmed cell death) instead of allowing the cell to replicate damaged genetic material.