Жасуша циклiн реттеушi кешенi (APC/C)

Cell cycle regulatory complex
the cell cycle regulatory complex, APC/C

Anaphase promoting complex (also called the cyclosome or APC/C) is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome. The APC/C is a large complex of 11–13 subunit proteins, including a cullin (Apc2) and RING (Apc11) subunit much like SCF. Other parts of the APC/C have unknown functions but are highly conserved. It was the discovery of the APC/C (and SCF) and their key role in eukaryotic cell cycle regulation that established the importance of ubiquitin mediated proteolysis in cell biology. Once perceived as a system exclusively involved in removing damaged protein from the cell, ubiquitination and subsequent protein degradation by the proteasome is now perceived as a universal regulatory mechanism for signal transduction whose importance approaches that of protein phosphorylation. In 2014, the APC/C was mapped in 3D at a resolution of less than a nanometre, which also uncovered its secondary structure. This finding could improve understanding of cancer and reveal new binding sites for future cancer drugs.

The APC/C's main function is to trigger the transition from metaphase to anaphase by tagging specific proteins for degradation. The three major targets for degradation by the APC/C are securin and S and M cyclins. Securin releases separase, a protease, when degraded. Separase then triggers the cleavage of cohesin, the protein complex that binds sister chromatids together. During metaphase, sister chromatids are linked by intact cohesin complexes. When securin undergoes ubiquitination by the APC/C and releases separase, which degrades cohesin, sister chromatids become free to move to opposite poles for anaphase. The APC/C also targets the mitotic cyclins for degradation, resulting in the inactivation of M CDK (mitotic cyclin dependent kinase) complexes, promoting exit from mitosis and cytokinesis. The critical substrates of the APC/C appear to be securin and the B type cyclins. This is conserved between mammals and yeast. In fact, yeast are viable in the absence of the APC/C if the requirement for targeting these two substrates is eliminated.

There is not a vast amount of extensive investigation on APC/C subunits, which serve mostly as adaptors. Studies of APC subunits are mainly conducted in yeast, and studies show that the majority of yeast APC subunits are also present in vertebrates, this suggests conservation across eukaryotes. Eleven core APC subunits have been found in vertebrates versus thirteen in yeast. The major motifs in APC subunits include tetratricopeptide (TPR) motifs and WD40 repeats 1. It is also suggested that variations in these WD40 domains result in varying substrate specificity, which is confirmed by recent results suggesting that different APC substrates can directly and specifically bind to Cdc20 and Cdh1/Hct1 Ultimately, the specificity differences are responsible for the timing of the destruction of several APC targets during mitosis. With CDC20 targeting a few major substrates at metaphase and Cdh1 targeting a broader range of substrates towards late mitosis and G1. Most notably, 4 subunits of yeast APC/C consist almost entirely of multiple repeats of the 34 amino acid tetratricopeptide residue (TPR) motif. These TPR subunits, Cdc16, Cdc27, Cdc23, and Apc5, mainly provide scaffolding and support to mediate other protein protein interactions. Cdc27 and Cdc23 have been shown to support the binding of Cdc20 and Cdh1, as mutations in key residues of these subunits led to increased dissociation of the activators. Apc10/Doc1, has been shown to promote substrate binding by mediating their interactions with Cdh1 and Cdc20. In particular, CDC20 (also known as p55CDC, Fizzy, or Slp1) inactivates CDK1 via ubiquitination of B type cyclins. This results in activation of Cdh1(a. k. a. Fizzy related, Hct1, Ste9, or Srw1), which interacts with APC during late mitosis and G1/G0. Cdh1 is inactivated via phosphorylation during S, G2 and early M phase. During these points in the cycle, it is not able to be assembled. Evidence shows that APC3 and APC7 serve to recruit Cdh1 to the anaphase promoting complex. This further supports that Cdh1 is responsible for maintaining APC activity during G1. Cdh1 does not require APC to be phosphorylated in order to bind, in fact, phosphorylation of Cdh1 by Cdks prevents it from binding to APC from S to M phase. With destruction of M Cdk, release of CDC20 from the APC and binding of Cdh1 can now occur, allowing APC activity to continue on during G1 entry.

One of the subunits that exhibit the TPR motif, CDC27 has been identified to interact with mitotic checkpoint proteins such as Mad2, p55CDC and BUBR1, suggesting that it may have involvement in the timing of M phase. Evidence shows that CDC27 is involved in a ternary complex with SMAD2/3 and Cdh1, which is created in response to TGFβ signalling. Because of its interaction with Cdh1 in particular, it has a potential role in determining affinity between APC and its activators Cdc20 and Cdh1. A study suggests that TGF β induced Cdc27 phosphorylation enhances interaction between cdc27 and Cdh1–which is directly involved in activating APC. CDC27 can serve as a vehicle through which TGFβ signalling can activate APC. Induced CDC27 hyperphosphorylation by TGFβ showed elevated APC activity.

CDC23, another TPR subunit interacts with SWM1, binding to the D box of CLB2. Based upon hybrid assays in vivo and co immunoprecipitation in vitro, it is suggested that Cdc16p, Cdc23p and Cdc27p (analogs in Sacchyromyces cerevisiae) interact and form a macromolecular complex. Their common theme of TPR is suggested to mediate these interactions. As for Cdc27 and Cdc16 in drosophila, their functions have been tested via RNA interference (RNAi). Results suggest that they may mediate activity of the entire complex via different mechanisms at different sites. In further drosophila studies, Cdk16 and cdk23 appear to be activated via phosphorylation by Polo like kinase 1 (Plk1) and its fission yeast counterpart, appear to bind particularly to Cdc23. The complex is understood to be regulated by activators CDC20 and Cdh1 during mitosis. Their role in degradation for cyclin B is demonstrated by a screen of Saccharomyces cerevisiae mutants defective for cyclin B degradation, which were found to have mutations in CDC16 and CDC23 genes. Mutants for CDC27, CDC23 and CDC 27 all resulted in a cell cycle arrest at metaphase.

APC/C substrates have recognition amino acid sequences that enable the APC/C to identify them. The most common sequence is known as the destruction box or D box. APC/C brings together an E2 ubiquitin conjugating enzyme and the D box rather than being an intermediate covalent carrier. The D box should have a version of the following amino acid sequence: RXXLXXXXN, where R is arginine, X is any amino acid, L is Leucine, and N is asparagine. The Ken box is another motif of importance. Its sequence should resemble the one that follows: KENXXXN, where K is lysine and E is glutamate. The last amino acid position in the Ken box is highly variable. Though it has been shown that mutations in the sequences do inhibit destruction of the proteins "in vivo", there is still much to learn about how proteins are targeted by the APC/C. Many APC substrates contain both D and KEN boxes, with their ubiquitylation by either APC/CCdc20 or APC/CCdh1 dependent on both sequences, yet some substrates contain only either a D box or a KEN box, in one or multiple copies. Having two distinct degradation sequences creates a high level of substrate specificity on the APC/C, with APC/CCdc20 being more dependent on the D box and APC/CCdh1 more dependent on the KEN box. For example, APC/CCdh1 is capable of ubiquitylating KEN box only containing substrates like Tome 1 and Sororin. From these early observations, it has been confirmed that in G2 and early mitosis, Emi1 binds and inhibits Cdc20 by preventing its association with APC substrates. Cdc20 can still be phosphorylated and bind to APC/C, but bound Emi1 blocks Cdc20's interaction with APC targets. Emi1's destruction leads APC/CCdc20 activation, allowing for the destruction of cyclin A in early mitosis. Emi1 levels begin to rise again in G, which help inhibit APC/CCdh1. Regulation of APC/CCdc20 activity towards metaphase substrates like securin and cyclin B may be a result of intracellular localization. It is hypothesized that spindle checkpoint proteins that inhibit APC/CCdc20 only associate with a subset of the Cdc20 population localized near the mitotic spindle. In this manner, cyclin A can be degraded while cyclin B and securin are degraded only once sister chromatids have achieved bi orientation.