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      The master cell cycle regulator APC-Cdc20 regulates ciliary length and disassembly of the primary cilium

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          Abstract

          The primary cilium has an important role in signaling; defects in structure are associated with a variety of human diseases. Much of the most basic biology of this organelle is poorly understood, even basic mechanisms, such as control of growth and resorption. We show that the activity of the anaphase-promoting complex (APC), an E3 that regulates the onset of anaphase, destabilizes axonemal microtubules in the primary cilium. Furthermore, the metaphase APC co-activator, Cdc20, is specifically recruited to the basal body of primary cilia. Inhibition of APC-Cdc20 activity increases the ciliary length, while overexpression of Cdc20 suppresses cilium formation. APC-Cdc20 activity is required for the timely resorption of the cilium after serum stimulation. In addition, APC regulates the stability of axonemal microtubules through targeting Nek1, the ciliary kinase, for proteolysis. These data demonstrate a novel function of APC beyond cell cycle control and implicate critical role of ubiquitin-mediated proteolysis in ciliary disassembly.

          DOI: http://dx.doi.org/10.7554/eLife.03083.001

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          The majority of cells in the human body have small hair-like structures that project from the cell surface. These structures, known as primary cilia, are involved in sensing light and touch, and they are also required for an organism to develop normally. Defects in cilia result in a wide range of human diseases that are collectively known as ciliopathies. These include polycystic kidney disease and Bardet–Biedl syndrome. Ciliary disorders can also affect almost every organ in the body leading to blindness, obesity, diabetes, and cancer.

          Cilia are dynamic structures that are dis-assembled when cells start to divide and are then re-assembled when cells are quiescent. The anaphase promoting complex (APC) has a critical role during cell division and targets key proteins that need to be degraded at specific times during this process. APC is localized in the basal body, which is found at the bottom of cilia, and it works together with a number of proteins which assist its function.

          Wang et al. now report that a complex formed by APC and its co-activator protein Cdc20 has two functions at the basal body: it is needed to maintain the optimal length of the cilia in quiescent cells and to shorten the cilia when cells exit from quiescent stage.

          Wang et al. also investigated the role of Nek1, an enzyme that is localised in the basal body. It was found that reducing the level of Nek1 in quiescent cells resulted in the formation of defective cilia, suggesting that this enzyme controls the stability and integrity of cilia. Moreover, when cells undergo division, the APC-Cdc20 complex targets the Nek1 enzyme, causing it to be degraded and allowing the cilia to be disassembled. A detailed understanding of how cells maintain the length of cilia could lead to the development of new approaches for the treatment of human ciliopathies.

          DOI: http://dx.doi.org/10.7554/eLife.03083.002

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          Most cited references29

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          The primary cilium at a glance.

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            The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1.

            The ordered progression through the cell cycle depends on regulating the abundance of several proteins through ubiquitin-mediated proteolysis. Degradation is precisely timed and specific. One key component of the degradation system, the anaphase promoting complex (APC), is a ubiquitin protein ligase. It is activated both during mitosis and late in mitosis/G(1), by the WD repeat proteins Cdc20 and Cdh1, respectively. These activators target distinct sets of substrates. Cdc20-APC requires a well-defined destruction box (D box), whereas Cdh1-APC confers a different and as yet unidentified specificity. We have determined the sequence specificity for Cdh1-APC using two assays, ubiquitination in a completely defined and purified system and degradation promoted by Cdh1-APC in Xenopus extracts. Cdc20 is itself a Cdh1-APC substrate. Vertebrate Cdc20 lacks a D box and therefore is recognized by Cdh1-APC through a different sequence. By analysis of Cdc20 as a substrate, we have identified a new recognition signal. This signal, composed of K-E-N, serves as a general targeting signal for Cdh1-APC. Like the D box, it is transposable to other proteins. Using the KEN box as a template, we have identified cell cycle genes Nek2 and B99 as additional Cdh1-APC substrates. Mutation in the KEN box stabilizes all three proteins against ubiquitination and degradation.
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              Pharmacologic inhibition of the anaphase-promoting complex induces a spindle checkpoint-dependent mitotic arrest in the absence of spindle damage.

              Microtubule inhibitors are important cancer drugs that induce mitotic arrest by activating the spindle assembly checkpoint (SAC), which, in turn, inhibits the ubiquitin ligase activity of the anaphase-promoting complex (APC). Here, we report a small molecule, tosyl-L-arginine methyl ester (TAME), which binds to the APC and prevents its activation by Cdc20 and Cdh1. A prodrug of TAME arrests cells in metaphase without perturbing the spindle, but nonetheless the arrest is dependent on the SAC. Metaphase arrest induced by a proteasome inhibitor is also SAC dependent, suggesting that APC-dependent proteolysis is required to inactivate the SAC. We propose that mutual antagonism between the APC and the SAC yields a positive feedback loop that amplifies the ability of TAME to induce mitotic arrest. Copyright © 2010 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: Reviewing editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                19 August 2014
                2014
                : 3
                : e03083
                Affiliations
                [1 ]Department of Systems Biology, Harvard Medical School , Boston, United States
                Stanford University , United States
                Stanford University , United States
                Author notes
                [* ]For correspondence: marc@ 123456hms.harvard.edu
                Article
                03083
                10.7554/eLife.03083
                4135350
                25139956
                a78ca39d-434e-49cd-b2e1-d6154542b5e6
                Copyright © 2014, Wang et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 05 May 2014
                : 28 July 2014
                Funding
                Funded by: National Institute of General Medical Sciences FundRef identification ID: http://dx.doi.org/10.13039/100000057
                Award ID: R01 GM039023
                Award Recipient :
                The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Cell Biology
                Custom metadata
                0.7
                The anaphase promoting complex (APC) has an essential role in ubiquitin-mediated proteolysis in the disassembly of cilia.

                Life sciences
                primary cilum,ubiquitin,cell cycle,retinal cell culture,none
                Life sciences
                primary cilum, ubiquitin, cell cycle, retinal cell culture, none

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