Cyclin E/Cdk2

The Cyclin E/Cdk2 complex is a structure composed of two proteins, cyclin E and cyclin-dependent kinase 2 (Cdk2). Similar to other cyclin/Cdk complexes, the cyclin E/Cdk2 dimer plays a crucial role in regulating the cell cycle, with this specific complex peaking in activity during the G1/S transition. Once the two cyclin and Cdk subunits are joined together, the complex becomes activated and proceeds to phosphorylate and bind to downstream proteins to ultimately promote cell cycle progression. Although cyclin E can bind to other Cdk proteins, its primary binding partner is Cdk2, and the majority of cyclin E activity occurs when it exists as the cyclin E/Cdk2 complex.

G1/S transition
Across eukaryotic cell types, the cell cycle is highly conserved, and the cyclin/Cdk complexes are consistently essential in driving the entire process forwards. Shortly before the end of G2 phase, cyclin E joins with Cdk2 to activate its serine-threonine kinase activity and thus promote entry into S phase. Eukaryotic cells possess two types of cyclin, cyclin E1 and cyclin E2, with the protein sequences sharing 69.3% similarity in humans despite being encoded by two different genes. While there is significant overlap in function between the two cyclin Es, there are distinct differences in specific roles and regulation for each cyclin E type. For example, in Xenopus laevis embryos only cyclin E1 is necessary for viability.

In living cells, over-expression (an excess amount) of either cyclin E type results in an earlier activation of the cyclin E/Cdk2 complex and the subsequent shortening of G1 phase and accelerated movement into S phase. The cyclin E/Cdk2 complex is not only important in regulating the G1/S transition, but in fact necessary, as cells lacking functional cyclin E are unable to enter S phase, remaining forever arrested in G1.

Complex activation
The cyclin E protein contains a section called the cyclin box, which interacts with the PSTAIRE helix on Cdk2 to enact a conformational change in Cdk2's T loop. The resulting exposure of Cdk2's catalytic site enables Cdk activating kinase (CAK) to phosphorylate Cdk2, allowing full activation of the cyclin E/Cdk2 complex. Once the protein dimer is formed and activated, it phosphorylates several important proteins including "proteins involved in centrosome duplication (NPM, CP110, Mps1), DNA synthesis (Cdt1), DNA repair (Brca1, Ku70), histone gene transcription (p220/NPAT, CBP/p300, HIRA) and Cdk inhibitors p21Waf1/Cip1 or p27Kip1." The complex interacts with its substrates due to two distinct regions of the cyclin E protein–the MRAIL and VDCLE domains. MRAIL is located at the N-terminus of cyclin E's cyclin box and interacts with proteins containing an RLX sequence (argininine-leucine-any amino acid) such as Rb, and p27KIP1. VDCLE is located at cyclin E's C-terminal region and interacts with proteins of the retinoblastoma family including Rb1, p107, and p130.

Localization
Cyclin E is predominantly found in the cell nucleus, but actually shuttles between the nucleus and the cytoplasm, however it typically appears as a nuclear protein when studied as its nuclear import is more rapid than its export. Cyclin E's nuclear localization sequence (NLS) allows the cyclin E/Cdk2 complex to readily enter the nucleus, although other mechanisms are believed to help the complex localize to the nucleus as well. Cyclin E also contains a centrosome localization sequence (CLS) that plays a key role in allowing the cyclin E/Cdk2 complex to control cetrosome duplication during early S phase.

Background–phosphorylation
The retinoblastoma tumor suppressor protein (Rb) plays a key regulatory role in several cellular activities, such as the G1 restriction checkpoint, the DNA damage checkpoint, cell cycle exit, and cellular differentiation. As its full name suggests, cells containing mutations in pathways upstream of Rb or in the protein itself (however this case is more rare), are often cancerous. In fact, the majority of human cancer cells contain mutations in proteins responsible for phosphorylating Rb, such as deletions (p16) or over-expressions (cyclin D, Cdk4, Cdk6).

Within its structure, Rb contains 16 possible sites for phosphorylation by other proteins. Surprisingly, however, it exists in only 3 possible states: un-phosphorylated (no sites phosphorylated), mono-phosphorylated (one site phosphorylated), or hyper-phosphorylated (all available sites phosphorylated). In G0 phase, Rb exists solely in its un-phosphorylated form, but in early G1 phase, the Cyclin D:Cdk4/6 complex adds one phosphate group and the protein remains in its mono-phosphorylated form until late G1 when it is rapidly hyper-phosporylated by the Cyclin E/Cdk 2 complex.

Cell cycle progression
The key mechanism through which the cyclin E/Cdk2 complex is able to promote S phase progression is through Rb and E2F transcription factors. Transcription factors (TF) regulate the rate at specific target genes are transcribed from DNA to RNA, i.e. transcription. At the end of G1, cells move through the restriction point–essentially "the point of no return" as cells that pass through are irreversibly committed to division and extracellular signals are no longer required for cell cycle progression. The rapid accumulation and activation of the cyclin E/Cdk 2 complex through positive feedback loops drives the cell forward through G1.

After phosphorylation by Cyclin D:Cdk4/6, mono-phosphorylated Rb binds to E2F family proteins, preventing their target genes from being transcribed; interestingly, one of said target genes is cyclin E. Through an unknown pathway, once a metabolic threshold is surpassed, a sensor triggers activation of the Cyclin E/Cdk2 complex leading to the hyper-phosphorylation of Rb. Mono-phosphorylated Rb inactivates E2F TFs, but hyper-phosphorylation of Rb inactivates it resulting in the release of E2F proteins and subsequent activation of E2F and transcription of its target genes. As a result, more cyclin E is transcribed and more of the cyclin E/Cdk2 complex is formed and activated. Cyclin E/Cdk2 is able to facilitate its own activation, leading to a rapid accumulation of the complex and simultaneous rapid hyper-phosphorylation (i.e. inactivation) of Rb leading to a switch-like transition through the restriction point into late G1 phase. In summary, cyclin E/Cdk2 inactivates Rb which activates E2F which activates more cylin E (and thus the cyclin E/Cdk2 complex), restarting the cycle and creating a positive feedback loop that results in sudden inactivation of Rb and push past the restriction point.