Methods to measure ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex

The anaphase-promoting complex (APC) or cyclosome is a multi-subunit ubiquitin ligase that controls progression through mitosis and the G1-phase of the cell cycle. The APC ubiquitinates regulatory proteins such as securin and cyclin B and thereby targets them for destruction by the 26S proteasome. Activation of the APC depends on the activator proteins Cdc20 and Cdh1, which are thought to recruit substrates to the APC. In vitro, APC's RING finger subunit Apc11 alone can also function as a ubiquitin ligase. Here, we review different methods that have been used to measure the ubiquitination activity of the APC in vitro and to analyze APC-mediated degradation reactions either in vitro or in vivo. We describe procedures to isolate the APC from human cells or from Xenopus eggs, to activate purified APC with recombinant Cdc20 or Cdh1 and to measure the ubiquitination activity of the resulting APC(Cdc20) and APC(Cdh1) complexes. We also describe procedures to analyze the ubiquitination activity associated with recombinant Apc11.     

Pseudosubstrate inhibition of the anaphase-promoting complex by Acm1: regulation by proteolysis and Cdc28 phosphorylation

The ubiquitin ligase activity of the anaphase-promoting complex (APC)/cyclosome needs to be tightly regulated for proper cell cycle progression. Substrates are recruited to the APC by the Cdc20 and Cdh1 accessory proteins. The Cdh1-APC interaction is inhibited through phosphorylation of Cdh1 by Cdc28, the major cyclin-dependent protein kinase in budding yeast. More recently, Acm1 was reported to be a Cdh1-binding and -inhibitory protein in budding yeast. We found that although Acm1 is an unstable protein and contains the KEN-box and D-box motifs typically found in APC substrates, Acm1 itself is not an APC substrate. Rather, it uses these motifs to compete with substrates for Cdh1 binding, thereby inhibiting their recruitment to the APC. Mutation of these motifs prevented Acm1-Cdh1 binding in vivo and rendered Acm1 inactive both in vitro and in vivo. Acm1 stability was critically dependent on phosphorylation by Cdc28, as Acm1 was destabilized following inhibition of Cdc28, mutation of consensus Cdc28 phosphorylation sites in Acm1, or deletion of the Bmh1 and Bmh2 phosphoprotein-binding proteins. Thus, Cdc28 serves dual roles in inhibiting Cdh1-dependent APC activity during the cell cycle: stabilization of the Cdh1 inhibitor Acm1 and direct phosphorylation of Cdh1 to prevent its association with the APC.     

Emi1 proteolysis: how SCF(beta-Trcp1) helps to activate the anaphase-promoting complex

Emi1 inhibits the anaphase-promoting complex (APC) during S and G2 phase. Two papers by Guardavaccaro et al. and Margottin-Goguet et al. in the June issue of Developmental Cell now show that Emi1 degradation in early mitosis is mediated by beta-Trcp1, an adaptor protein that recruits proteins to the SCF ubiquitin ligase.     

The END network couples spindle pole assembly to inhibition of the anaphase-promoting complex/cyclosome in early mitosis

Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the anaphase-promoting complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear. Here we describe a critical spindle pole-associated mechanism, called the END (Emi1/NuMA/dynein-dynactin) network, that spatially restricts APC/C activity in early mitosis. The APC/C inhibitor Emi1 binds the spindle-organizing NuMA/dynein-dynactin complex to anchor and inhibit the APC/C at spindle poles, and thereby limits destruction of spindle-associated cyclin B. Cyclin B/Cdk1 activity recruits the END network and establishes a positive feedback loop to stabilize spindle-associated cyclin B critical for spindle assembly. The organization of the APC/C on the spindle also provides a framework for understanding microtubule-dependent organization of protein destruction.     

The dephosphorylated form of the anaphase-promoting complex protein Cdc27/Apc3 concentrates on kinetochores and chromosome arms in mitosis

Cell cycle regulated protein ubiquitination and degradation within subcellular domains may be essential for the normal progression of mitosis. Cdc27 is a conserved component of an essential M-phase ubiquitin-protein ligase called the anaphase-promoting complex/cyclosome. We examined the subcellular distribution of Cdc27 in greater detail in mammalian cells and found Cdc27 concentrated at spindle poles and on spindle microtubules as previously described, but also found Cdc27 at kinetochores and along chromosome arms. This localization was not dependent on intact microtubules. While the great majority of Cdc27 protein in M phase cells is highly phosphorylated, only the dephosphorylated form of Cdc27 was found associated with isolated chromosomes. Kinases that also associate with isolated chromosomes catalyzed the in vitro phosphorylation of the chromosome-associated Cdc27. Microinjection of anti-Cdc27 antibody into cells causes arrest at metaphase. Microinjection of cells with anti-Mad2 antibody normally induces premature anaphase onset resulting in catastrophic nondisjunction of the chromosomes. However, coinjection of anti-Cdc27 antibody with anti-Mad2 antibody resulted in metaphase arrest. The association of dephosphorylated APC/C components with mitotic chromosomes suggests mechanisms by which the spindle checkpoint may regulate APC/C activity at mitosis.     

Subunits and substrates of the anaphase-promoting complex

The initiation of anaphase and exit from mitosis depend on a ubiquitination complex called the anaphase-promoting complex (APC) or cyclosome. The APC is composed of more than 10 constitutive subunits and associates with additional regulatory factors in mitosis and during the G1 phase of the cell cycle. At the metaphase-anaphase transition the APC ubiquitinates proteins such as Pds1 in budding yeast and Cut2 in fission yeast whose subsequent degradation by the 26S proteasome is essential for the initiation of sister chromatid separation. Later in anaphase and telophase the APC promotes the inactivation of the mitotic cyclin-dependent protein kinase 1 by ubiquitinating its activating subunit cyclin B. The APC also mediates the ubiquitin-dependent proteolysis of several other mitotic regulators, including other protein kinases, APC activators, spindle-associated proteins, and inhibitors of DNA replication.     

The gamma-secretase complex: machinery for intramembrane proteolysis

Gamma-secretase is a membrane protease complex that possesses presenilin as a catalytic subunit. Presenilin generates amyloid beta peptides in the brains of Alzheimer's patients and is indispensable to Notch signaling in tissue development and renewal. Recent studies have revealed how presenilin is assembled with its cofactor proteins and acquires the gamma-secretase activity: Aph-1 and nicastrin initially form a subcomplex to bind and stabilize presenilin, and then Pen-2 confers the gamma-secretase activity and facilitates endoproteolysis of presenilin. Understanding the mechanism of gamma-secretase cleavage will help to clarify how intercellular cell signaling through transmembrane proteins is regulated by intramembrane proteolysis, and will hopefully eventually lead to a cure for Alzheimer's disease.     

Three-dimensional structure of the anaphase-promoting complex

The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-protein ligase, composed of at least 11 subunits, that controls progression through mitosis and G1. Using cryo-electron microscopy and angular reconstitution, we have obtained a three-dimensional model of the human APC at a resolution of 24 A. The APC has a complex asymmetric structure 140 A x 140 A x 135 A in size, in which an outer protein wall surrounds a large inner cavity. We discuss the possibility that this cavity represents a reaction chamber in which ubiquitination reactions take place, analogous to the inner cavities formed by other protein machines such as the 26S proteasome and chaperone complexes. This cage hypothesis could help to explain the great subunit complexity of the APC.     

The chromosomal passenger complex: guiding Aurora-B through mitosis

During mitosis, the chromosomal passenger complex (CPC) orchestrates highly different processes, such as chromosome alignment, histone modification, and cytokinesis. Proper and timely localization of this complex is the key to precise control over the enzymatic core of the CPC, the Aurora-B kinase. We discuss the molecular mechanisms by which the CPC members direct the dynamic localization of the complex throughout cell division. Also, we summarize posttranslational modifications that occur on the CPC and discuss their roles in regulating localization and function of this mitotic complex.     

Novel functions for the anaphase-promoting complex in neurobiology

In recent years, diverse and unexpected neurobiological functions have been uncovered for the major cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex (APC). Functions of the APC in the nervous system range from orchestrating neuronal morphogenesis and synapse development to the regulation of neuronal differentiation, survival, and metabolism. The APC acts together with the coactivating proteins Cdh1 and Cdc20 in neural cells to target specific substrates for ubiquitination and consequent degradation by the proteasome. As we continue to unravel APC functions and mechanisms in neurobiology, these studies should advance our understanding of the molecular mechanisms of neuronal connectivity, with important implications for the study of brain development and disease.     

Structural insights into anaphase-promoting complex function and mechanism

The anaphase-promoting complex or cyclosome (APC/C) controls sister chromatid segregation and the exit from mitosis by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. This unusually large E3 RING-cullin ubiquitin ligase is assembled from 13 different proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D box and KEN box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. Recent structural and biochemical studies of the APC/C are beginning to reveal an understanding of the roles of individual APC/C subunits and co-activators and how they mutually interact to mediate APC/C functions. This review focuses on the findings showing how information on the structural organization of the APC/C provides insights into the role of co-activators and core APC/C subunits in mediating substrate recognition. Mechanisms of regulating and modulating substrate recognition are discussed in the context of controlling the binding of the co-activator to the APC/C, and the accessibility and conformation of the co-activator when bound to the APC/C.     

The substrates of Plk1, beyond the functions in mitosis

Polo-like kinase 1 (Plk1) is a key regulator of cell division in eukaryotic cells. In this short review, we briefly summarized the well-established functions modulated by Plk1 during mitosis. Beyond mitosis, we focused mainly on the unexpected processes in which Plk1 emerges as a critical player, including microtubule dynamics, DNA replication, chromosome dynamics, p53 regulation, and recovery from the G2 DNA-damage checkpoint. Our discussion is mainly based on the critical substrates targeted by Plk1 during these cellular events and the functional significance associated with each phosphorylation event.     

The nuclear pore complex: nucleocytoplasmic transport and beyond

Over the past two years, it has become evident that there is an unexpected link between nuclear pore complex structure and dynamics, nucleocytoplasmic transport and chromosome segregation. In addition, a tomographic three-dimensional reconstruction of native nuclear pore complexes preserved in thick amorphous ice has unveiled a number of new structural features of this supramolecular machine. These data, together with some of the elementary physical principles that underlie nucleocytoplasmic transport, will be discussed in this review.     

The anaphase-promoting complex and separin are required for embryonic anterior-posterior axis formation

Polarization of the one-cell C. elegans embryo establishes the animal's anterior-posterior (a-p) axis. We have identified reduction-of-function anaphase-promoting complex (APC) mutations that eliminate a-p polarity. We also demonstrate that the APC activator cdc20 is required for polarity. The APC excludes PAR-3 from the posterior cortex, allowing PAR-2 to accumulate there. The APC is also required for tight cortical association and posterior movement of the paternal pronucleus and its associated centrosome. Depletion of the protease separin, a downstream target of the APC, causes similar pronuclear and a-p polarity defects. We propose that the APC/separin pathway promotes close association of the centrosome with the cortex, which in turn excludes PAR-3 from the posterior pole early in a-p axis formation.     

The anaphase-promoting complex is required in both dividing and quiescent cells during zebrafish development

The anaphase-promoting complex/cyclosome (APC/C) regulates multiple stages of the cell cycle, most prominently mitosis. We describe zebrafish with mutations in two APC/C subunits, Cdc16 and Cdc26, whose phenotypes reveal a multifaceted set of defects resulting from the gradual depletion of the APC/C. First, loss of the APC/C in dividing cells results in mitotic arrest, followed by apoptosis. This defect becomes detectable in different organs at different larval ages, because the subunits of the APC/C are maternally deposited, are unusually stable, and are depleted at uneven rates in different tissues. Second, loss of the APC/C in quiescent or differentiated cells results in improper re-entry into the cell cycle, again in an apparently tissue-specific manner. This study is the first demonstration of both functions of the APC/C in a vertebrate organism and also provides an illustration of the surprisingly complex effects that essential, maternally supplied factors can have on the growing animal over a period of 10 days or longer.     

Destruction with a box: substrate recognition by the anaphase-promoting complex

Destruction boxes mark cyclin B and other proteins degraded in mitosis for ubiquitination by the anaphase-promoting complex (APC/C). In a paper in this issue of Molecular Cell, Yamano et al. show that destruction boxes directly bind to the APC/C in a cell cycle-regulated manner. Interestingly, this interaction does not require APC/C activators of the Cdc20 family, which were thought to be essential for recruiting substrates to the APC/C.     

Substrate-specific regulation of ubiquitination by the anaphase-promoting complex

By orchestrating the sequential degradation of a large number of cell cycle regulators, the ubiquitin ligase anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The correct timing of APC/C-dependent substrate degradation, a critical feature of progression through mitosis, was long known to be controlled by mechanisms targeting the core APC/C-machinery. Recent experiments, however have revealed an important contribution of substrate-specific regulation of the APC/C to achieve accurate cell division. In this perspective, we describe different mechanisms of substrate-specific APC/C-regulation and discuss their importance for cell division.Key words: ubiquitin, proteasome, anaphase-promoting complex, spindle assembly factors, degradation