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APC/C Signaling Pathway


Figure 1. APC/C signaling pathway.

APC/C overview

The anaphase-promoting complex/cyclosome (APC/C) is a multifunctional ubiquitin ligase involved in cell cycle, metabolism, DNA damage repair, autophagy, apoptosis, aging, tumors and a variety of biological processes. As an important post-translational modification, ubiquitination regulates protein degradation by the ubiquitin-proteasome system (UPS). APC/C has a large molecular weight and consists of multiple subunits. It plays an important role in cell cycle regulation and can precisely regulate cell cycle transition by mediating ubiquitination of cell cycle-associated proteins and is co-activated by CDC20 or regulation of CDH1. Understanding the structure and function of APC/C is critical for studying biological events such as cell cycle and post-translational modification of proteins. In recent years, great progress has been made in the analysis of the structure and composition of APC/C molecules, and its role in tumors and potential therapeutic applications have also received attention.

APC/C family

APC/C is a giant multi-subunit protein complex having a molecular weight of about 1.2 MD. In mammals, its core is composed of at least 19 subunits. In addition to the core, APC/C binds to the co-activator molecule CDC20 or CDH1, which is responsible for the attachment of substrates and regulation of APC/C activity. According to a recent analysis of the molecular structure of APC/C by Barford's research group, APC/C can be divided into three sub-complexes, which are the scarffolding, the catalytic and substrate recognition core and the TPR arm containing a peptide repeat domain. The scaffold subcomplex platform includes APC1, APC4, and APC5, and the catalytic and substrate recognition cores include APC2, APC10, APC11, and CDC20/CDH1. The TPR arm includes APC3, APC6, APC7, APC8, APC12, APC13, and APC16. Among the individual subunits of APC/C, APC3, APC6, APC7, APC8, and APC12 exist in a dimer form, while other subunits exist in a monomeric form. APC1 is linked to the catalytic core and the TPR arm; APC3 is a catalytic core scaffold that recognizes the homologous C-terminal Ile-Arg sequence of APC10 and CDC20/CDH1, thereby recruiting CDC20 or CDH1; APC2-APC11 regulates APC/C and E2 interaction; APC10 is the most important site for binding to the substrate and can participate in the recruitment of substrates through its degron-recognition module. The TPR arm is an important scaffold for APC/C, in which APC8 provides a binding site for the scaffold subcomplex and co-activator (CDC20 or CDH1); APC12, APC13, and APC16 bind to the corresponding sites of the four dimer subunits of TRP, which may regulate the stability of the TPR arm; APC16 asymmetrically binds to the APC3 dimer, and can recruit APC7 to APC3 to participate in the assembly of the TPR arm; APC15 is not an essential molecule for APC/C involved in substrate ubiquitination, but participates in the negative regulation of CDC20/CDH1 and the initiation of mitosis. Although the current research on the structure of APC/C has made great progress, many specific issues still need further research, such as whether the APC/C function requires all subunits, whether the combination of different subunits can play different APC/C functions, and whether the function of the subunit of the scaffold section has specific regulatory functions.

APC/C signaling pathway

  1. APC/C signaling pathway cascade
  2. The progression of the normal cell cycle requires that certain cell cycle regulatory molecules rapidly degrade at specific times to complete DNA replication, progression of mitosis, maintenance of G1 phase, and cytokinesis. This precisely regulated degradation process is accomplished by APC/C catalyzed ubiquitination. APC/C regulates the cell cycle primarily through collaboration with CDC20 and CDH1. The APC/C-CDC20 complex primarily regulates the degradation of related substrates in the mid-mitotic stages. The APC/C-CDH1 complex primarily regulates the progression of late mitosis and G1 phase. CDC20 plays an important role in the G2 phase of progress. In the G2 phase, CDC20 is phosphorylated by a kinase such as CDK1 (cyclin-dependent kinase 1), partially activating its interaction with APC/C. At the G2/M phase transition, the activity of the APC/C-CDC20 complex is inhibited. In the middle stage of mitosis, the APC/C-CDC20 complex can ubiquitinate and degrade Cyclin A, and NEK2A (NIMA-related expressed kinase 2A). The active Cyclin B1-CDK1 complex maintains the cells in the M phase, giving the spindle enough time to properly link to the chromosome. Securin inhibits the segregation of sister chromatids. To complete cell division, cell Cycle B1 and Securin must be degraded later. After passing through the cell cycle checkpoint, inhibition of the APC/C-CDC20 complex disappears, and Securin and Cyclin B1 are degraded in the metaphase. Cyclin B3 enhances APC/C activity and promotes metaphase to late conversion. CDH1 is ubiquitous in the cell cycle but is active from the end of mitosis to the G1 phase. Due to phosphorylation, CDH1 is inactive during both G2 and early mitosis. CDH1 has only partial activity in the middle of mitosis, which can mediate the degradation of Securin and CDC20 in the presence of SAC and promote the formation of APC/C-CDH1 complex. Aurora B acts as a component of the chromosomal shift complex and regulates chromosome segregation and cytokinesis; Aurora A has a wide range of functions, such as interaction with TPX2 (targeting protein for Xenopus kinesin-like protein 2) to mediate spindle assembly; PLK1 (Polo -like kinase 1) participates in centrosome microtubule nucleation, spindle polarity establishment and cytokinesis. In the late stage of mitosis, CDH1 is dephosphorylated by CDC14, activating the APC/C-CDH1 complex. In the late stages, ubiquitination degrades CDC20, Aurora A, Aurora B, PLK1 and other kinases to promote the end of the mitotic phase. In the G1 phase, the APC/C-CDH1 complex degrades mitosis Cyclin, CDC25A, SKP2 (S-phase kinase-associated protein 2), USP37 (ubiquitin specific peptidase 37), etc., which reduces CDK activity and degrades CDC6. Replication regulatory proteins such as RRM2 (ribonucleotide reductase regulatory subunit M2), CLASPIN, etc., cause their own inactivation and maintain the content of Cyclin A. In the G1/S phase transition, the APC/C-CDH1 complex is inactivated due to degradation of CDH1 and E2 and inhibition by EMI1 (early mitotic inhibitor 1). In the S phase and G2 /S phase, the Cyclin A2-CDK2 complex binds to and phosphorylates CDH1 to maintain the inactivation of APC/C and prevent its degradation of substrates such as Cyclin A2 and Cyclin B1. The cascade reaction through E1, E2, and E3 is the main mechanism for ubiquitination modification of substrate proteins. The substrates of APC/C contain short linear recognition sequences, such as the D-[RXXLXXXX(N)] box and the KEN-[KENXXX(N)] box, which can only be ubiquitinated by polyubiquitination. Monoubiquitination regulates the localization, stability, and activity of substrate proteins, while polyubiquitin chains formed at different sites of ubiquitin molecules play different functions. Currently, APC/C is known to form polyubiquitin chains through the lysine 11, 48 and 63. However, recent studies have shown that Lys 11 is the major polyubiquitination site in cell cycle-associated APC/C activity. E2 involved in APC/C-mediated ubiquitination is mainly divided into two categories: UBCH10 or UBCH5, which is responsible for the initiation of APC/C ubiquitination, catalyzing the attachment of the first ubiquitin molecule to the substrate; UBE2S catalyzing the ubiquitin chain extends and forms ubiquitin chain branches. The process of forming the Lys 11 polyubiquitin chain under the action of UBCH10 and UBE2S is as follows: First, UBCH10 is recruited to APC/C, and the substrate is anchored by interaction with APC11 and APC2, and the substrate passes through the D-box. Combined with the KEN-box on APC/C and CDC20/CDH1. The interaction of the UBC domain of ubiquitin with the RING domain of APC11 activates UBCH10, allowing UBCH10 to act as a mediator and transfer ubiquitin to the substrate. Thereafter, CDC20/CDH1 recognizes UBE2S and exchanges UBCH10 from APC/C. APC11 assists in the attachment of ubiquitin on UBE2S to the first ubiquitin molecule on the substrate to form a polyubiquitin chain. The extension of the polyubiquitin chain can be inhibited by acetylation of ubiquitin.

  3. Pathway regulation
  4. The activity of APC/C is regulated by the coactivator molecule CDC20/CDH1. During cell cycle progression, APC/C can also be regulated by phosphorylation of the subunit, mitotic checkpoint complex (MCC), and EMI1. CDC20/CDH1: Both CDC20 and CDH1 contain a WD40 domain to recruit and bind substrates. They enhance the interaction between E2 and APC/C and can also be combined with APC3 and APC8 of the TPR arm structure. CDC20 binds to APC/C early in mitosis and degrades mitotic-related substrates. Next, CDH1 replaces CDC20, regulating late mitosis and G1. Subsequently, CDK1 phosphorylates CDH1 and inhibits the interaction of CDH1-APC/C. As CDK1 activity decreases, CDH1 dephosphorylates, restarting APC/C activity and mediating substrate degradation in the next cell cycle. Acetylation of CDC20 and CDH1 also inhibits APC/C activity. It has recently been discovered that CDC20 and CDH1 can also assist another small molecule, E3-PARKIN, to promote the degradation of some cyclins. Although the substrate of APC/C partially overlaps with the substrate of PARKIN, APC/C still dominates. MCC: The MCC includes the spindle assembly checkpoint (SAC) protein and CDC20, which prevents cells from beginning mitosis in the unprepared condition by inhibiting APC/C activity. After the chromosome is attached to the microtubule, APC/C immediately anchors Cyclin B and Securin. If the centromere is not attached to the microtubule, SAC will generate a “waiting” signal to inhibit APC/C degradation. Cyclin B and Securin catalyze the incorporation of a second CDC20 into the MCC, inhibiting APC/C that has bound and activated with CDC20. EMI1: EMI1 is an APC/C inhibitory protein that binds directly to the core complex of APC/C via a ZBR (zinc binding region) motif during mitosis and binds to CDC20/CDH1 via D-box, preventing APC/C recruitment. EMI2 competitively binds to the binding site of UBE2S to APC10, thereby inhibiting APC/C function. Phosphorylation of phosphorylated APC/C subunits is important for the regulation of APC/C activity. During the mitosis phase, most of the subunits of APC/C are phosphorylated at multiple sites, especially the multi-site phosphorylation of CDC20 and CDH1, which is part of a complex regulatory system. To prevent chromosomal segregation, CDC20 binds only after mitotic APC/C phosphorylation and is inhibited by MCC until all chromosomes are properly ligated to the spindle to activate APC/C function. Phosphorylation of CDH1 prevents its binding to APC/C. Phosphorylation of APC1 and APC3 is required for CDC20 to activate APC/C, and phosphorylation of APC12 regulates the assembly of TPR arm subunits, thereby modulating APC/C activity.

  5. Relationship with diseases
  6. Tumor

    The dysregulation of E3 ligase can lead to accumulation of oncogene products in cells or excessive degradation of tumor suppressor proteins, and APC/C-regulated cellular events directly or indirectly affect tumorigenesis and development. With the help of the transgenic mouse model, APC/C has a certain research effect on the development of tumorigenesis, mainly focusing on the relationship between APC/C core subunits, co-activators and tumors.

References

  1. Chang L, Zhang Z, Yang J, et al. Atomic structure of the APC/C and its mechanism of protein ubiquitination. Nature. 2015, 522(7557):450-4.
  2. Izawa D, Pines J. The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C. Nature. 2015, 517(7536):631-4.
  3. Zhang J, Wan L, Dai X, et al. Functional characterization of Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligases in tumorigenesis. Biochimica Et Biophysica Acta. 2014, 1845(2):277-93.
  4. Min M, Mevissen T E, De L M, et al. Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages. Molecular Biology of the Cell. 2015, 26(24):4325.
  5. Arnold L, Seufert W. Insights into the cellular mechanism of the yeast ubiquitin ligase APC/C-Cdh1 from the analysis of in vivo degrons. Molecular Biology of the Cell. 2015, 26(5):843-858.

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