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Mitotic Regulation in Higher Eukaryotes by Ran and SUMO-1
- Mary Dasso, PhD, Head, Section on Cell Cycle Regulation
- Maia Ouspenskaia, DVM, Biologist
- Kara Lukasiewicz, PhD, Postdoctoral Fellow
- Alexei Arnaoutov, PhD, Visiting Fellow
- Maiko Furuta, PhD, Visiting Fellow
- Ming-Ta Lee, PhD, Visiting Fellow
- Maria Lyanguzova, PhD, Visiting Fellow
- Sarine Markossian, PhD, Visiting Fellow
- Min Mo, PhD, Visiting Fellow
- Hyunju Ryu, PhD, Visiting Fellow
- Shaofei Zhang, BA, Graduate Student
We are interested in mechanisms of chromosome segregation. Defects in chromosome segregation lead to aneuploidy, the condition of an abnormal number of chromosomes, which in turn may drive tumor formation. To elucidate these mechanisms, we study the mitotic roles of proteins associated with the interphase nuclear pore complex (NPC). NPCs are conduits for nucleocytoplasmic trafficking and organize other nuclear processes, such as gene expression and the repair of DNA damage. Surprisingly, many NPC components (nucleoporins) localize to mitotic kinetochores, the proteinaceous structures that attach sister chromatids to mitotic spindles and mediate their segregation into daughter cells at anaphase. The Ran GTPase and SUMO conjugation pathways are functionally and physically linked to each other and to NPCs. The Ran GTPase controls many cellular activities, including nucleocytoplasmic trafficking, nuclear assembly, and cell cycle progression. SUMO (small ubiquitin-like modifier) proteins are a family of ubiquitin-like proteins that become covalently conjugated to cellular targets. The pathways are indispensable for mitotic spindle assembly and kinetochore function. The ultimate goal of our studies is to understand the mitotic roles of these proteins, discover how they are coordinated, and determine how such coordination enhances the accurate distribution of chromosomes during mitosis.
Mitotic roles of nuclear pore complex proteins
Trafficking between the nucleus and cytoplasm occurs through NPCs. Kinetochores are proteinaceous structures that assemble at the centromere of each sister chromatid during mitosis and serve as sites of spindle microtubule attachment. The relationship between mitotic kinetochores and NPCs is both surprisingly intimate and poorly understood. NPCs consist of around thirty proteins, called nucleoporins. During interphase, several kinetochore proteins also bind stably to NPCs (e.g., Mad1, Mad2, Mps1). During mitosis, metazoan NPCs disassemble, and at least a third of nucleoporins associate with kinetochores, including the RanBP2 complex and the Nup107-160 complex. We showed that these complexes play important roles in kinetochore function. Several other nucleoporins that do not associate with kinetochores have also been shown by others to play important mitotic roles, including Nup214, Nup98, and TPR. Much of our work has focused on two complexes of kinetochore-bound nucleoporins.
The RanBP2 complex consists of RanBP2 (a large nucleoporin that is also known as Nup358), SUMO-1–conjugated RanGAP1 (the activating protein for the Ran GTPase), and Ubc9 (the conjugating enzyme for the SUMO family of ubiquitin-like modifiers). The complex associates with kinetochores in a microtubule-dependent manner and also requires Crm1, a Ran-dependent nuclear export receptor. Disruption of RanBP2 association to kinetochores results in defective mitotic spindle assembly. Additional observations suggest an in vivo role of RanBP2 in interphase microtubule organization. Our current studies focus on interacting proteins that may be essential for the function of the RanBP2 complex.
The vertebrate Nup107–160 complex consists of ten nucleoporins. It is broadly distributed on spindles during prometaphase and remains kinetochore-bound throughout mitosis. Unattached kinetochores nucleate microtubules in a Ran-regulated manner; such microtubules promote assembly of kinetochore fibers (k-fibers), which connect kinetochores to spindle poles. We found that Nup107–160 interacts with the gamma-tubulin ring complex (gamma-TuRC), an essential and conserved microtubule nucleator, and that the association recruits gamma-TuRC to unattached kinetochores. The findings suggest a novel way in which Ran can influence mitotic spindle assembly. We are currently investigating the mechanism through which Ran regulates microtubule nucleation of gamma–TuRC associated with Nup107–160 at kinetochores.
Regulation of mitotic kinetochores by the Ran GTPase
The Ran GTPase is required for many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly, and cell cycle control. During interphase, the distribution of Ran regulators leads to a high concentration of Ran-GTP in nuclei and low Ran-GTP in cytosol. The major effectors for Ran are a family of Ran-GTP–binding proteins collectively known as karyopherins. Karyopherins that mediate nuclear import are called importins, and those that mediate export are called exportins. To date, two karyopherins, importin-beta and the exportin Crm1, have been shown to act as Ran effectors during mitosis.
Our studies have been particularly concerned with mitotic Ran functions at kinetochores. Kinetochore attachment is monitored through the spindle assembly checkpoint (SAC), which prevents mitotic exit until all chromosomes are attached and aligned onto the metaphase plate. Elevated levels of Ran-GTP abrogate SAC–mediated mitotic arrest in Xenopus egg extracts (XEEs) and disrupt the kinetochore localization of SAC components, suggesting that the SAC is directly responsive to the overall concentration of Ran-GTP in that system. The nature of the effector for Ran in the SAC remains an unresolved issue, and this problem is a major focus of our current interests. Our findings indicate that the effector is neither importin-beta nor Crm1.
Crm1 is the exportin for proteins with classical export signals (NESs). We found that Crm1 localizes to kintochores and that inhibition of Crm1 by Leptomycin B (LMB) in mitosis results in abnormal kinetochore attachment and reduced microtubule (MT) stability and spindle size. The defects are correlated with a failure to recruit the Ran–BP2 complex onto kinetochores after MT attachment is established. We examined the proteins that bind to Crm1, in order to determine whether Crm1 regulates target proteins through sequestration, as has been reported for importin-beta, and to ascertain whether any targets bind to Crm1 in a mitosis-specific fashion. We found a small set of sequestered cargos (SCs) that bind quantitatively to Crm1 in a Ran-GTP–dependent manner. This unusual property of SCs suggests that they are regulated by Crm1, but Crm1's role in controlling these targets appears to go beyond simply moving them out of the nucleus.
We identified a putative histone H3 lysine demethylase (KDM3B) as a SC and subjected it to further study. We examined the localization of KDM3B and found that it co-localizes with Crm1 in the nucleoplasm and in intranucleolar bodies (INBs). Strikingly, treatment with LMB caused rapid and complete loss of KDM3B from INBs, indicating that Crm1 is required for KDM3B localization at that site. Cells treated with Actinomycin D, an RNA polymerase I inhibitor, also lost KDM3B from INBs. Chromatin IP (ChIP) assays suggest that KDM3B binds to rRNA genes, and qPCR data reveal that treatment with LMB lowers transcription of rRNA genes. Collectively, the data suggest that interaction with Crm1–Ran-GTP regulates KDM3B–mediated histone demethylation and rRNA gene transcription in a novel, transport-independent manner.
SUMO–family small ubiquitin-like modifiers in higher eukaryotes
SUMOs are ubiquitin-like proteins (Ubls) that become conjugated to substrates through a pathway that is biochemically similar to ubiquitination. SUMOylation is involved in many cellular processes, including DNA metabolism, gene expression, and cell cycle progression. Vertebrate cells express three major SUMO paralogues (SUMO-1–3). Mature SUMO-2 and SUMO-3 are 95% identical to each other, while SUMO-1 is 45% identical to SUMO-2 or SUMO-3. Where they are functionally indistinguishable, SUMO-2 and SUMO-3 will be collectively referred to as SUMO-2/3. Like ubiquitin, SUMO-2/3 can be assembled into polymeric chains through the sequential conjugation of SUMOs to each other. A large number of SUMOylation substrates have been identified. SUMOylation promotes a variety of fates for individual targets, dependent upon the protein itself, the conjugated paralogue, and whether the conjugated species contains a single SUMO or SUMO chains.
SUMOylation is dynamic owing to rapid turnover of conjugated species by SUMO proteases. Both post-translational processing of SUMO polypeptides and deSUMOylation are mediated by the same family of proteases, which play a pivotal role in determining the spectrum of SUMOylated species. This group of proteases is called Ubl–specific proteases (Ulp) in yeast and Sentrin-specific proteases (SENP) in vertebrates. There are two yeast Ulps (Ulp1p and Ulp2p/Smt4p), and six mammalian SENPs (SENP1, 2, 3, 5, 6, and 7). SENP1, 2, 3, and 5 form a Ulp1p–related sub-family, while SENP6 and SENP7 are more closely related to Ulp2p. Yeast Ulps have important roles in mitotic progression and chromosome segregation. We defined the enzymatic specificity of the vertebrate SENP proteins and analyzed their key biological roles.
Ulp1p localizes to NPCs, is encoded by an essential gene, and is important for SUMO processing, nucleocytoplasmic trafficking, and late steps in the ribosome biogenesis pathway. While humans possess two NPC–associated SENPs, SENP1 and SENP2, we found that frogs possess a single member of this family, xSENP1. We are currently exploiting this difference to analyze the function of NPC–bound SUMO proteases. We determined the interaction partners of this enzyme throughout the cell cycle, using Xenopus egg extracts (XEEs), and are using XEEs to analyze both the function of xSENP1 and the role of its interacting partners.
We demonstrated a role of the Ulp1p–like protease SENP3 in ribosome biogenesis. Ribosome biogenesis is a major metabolic expense and a critical point of cellular regulation during both cell growth and cancer progression. The B23/NPM protein is essential for ribosome biogenesis and other cellular functions and is frequently mutated in human hematopoietic malignancies. We found that SENP3 binds stably to a conserved set of ribosome assembly factors (Rix1 complex) and that SENP3 and the Rix1 complex require B23/NPM for ribosome binding. Notably, the binding also requires high levels of Ran-GTP, a small nuclear GTPase that controls the direction of nuclear trafficking. Under low Ran-GTP conditions, B23/NPM was excluded from the complex and the importin RanBP5 bound instead. Our findings not only reveal conservation between the ribosome biogenesis pathways in yeast and vertebrates but also demonstrate a novel mechanism through which the vertebrate pathway is regulated by Ran.
Yeast Ulp2p is nucleoplasmic and not essential for vegetative growth but is important for chromosome segregation. Ulp2p acts particularly in disassembly of poly–SUMO chains. We demonstrated that human SENP6 is a Ulp2p–related vertebrate enzyme that similarly prefers substrates containing multiple SUMO-2/3 moieties. We analyzed the mitotic role of SENP6 and found that it is essential for accurate chromosome segregation. Mitotic defects observed in the absence of SENP6 reflected the loss of inner kinetochore proteins, including components of the CENP-H/I/K and CENP-O complexes. The findings demonstrate a novel function of the SUMO pathway in inner kinetochore assembly, which finely balances the incorporation with degradation of components of the inner plate. We are currently analyzing other aspects of SENP6 mitotic function, including its role in chromosome morphology.
- Pharmacology Research and Training Award (PRAT Fellowship) to Kara Lukasiewicz 10/2011–10/2012
- Mukhopadhyay D, Arnaoutov A, Dasso M. The SUMO protease SENP6 is essential for inner kinetochore assembly. J Cell Biol 2010;188:681-692.
- Ryu HJ, Al-Ani G, Deckert K, Kirkpatrick D, Gygi S, Dasso M, Azuma Y. PIASy mediates SUMO-2/3 conjugation of poly (ADP-ribose) polymerase 1 (PARP1) on mitotic chromosomes in vertebrates. J Biol Chem 2010;285:14415-14423.
- Mishra RK, Chakraborty P, Arnaoutov A, Fontoura BMA, Dasso M. The Nup107-160 complex and γ-TuRC regulate microtubule polymerization at kinetochores. Nat Cell Biol 2010;12:164-169.
- Bernad R, Sánchez P, Rivera T, Rodríguez-Corsino M, Boyarchuk E, Vassias I, Ray-Gallet D, Arnaoutov A, Dasso M, Almouzni G, Losada A. Xenopus HJURP and condensin II are required for CENP-A assembly. J Cell Biol 2011;192:569-582.
- Chow KH, Elgort S, Dasso M, Ullman KS. Two distinct sites in Nup153 mediate interaction with the SUMO proteases SENP1 and SENP2. Nucleus 2012;3:349-358.
- Yoshiaki Azuma, PhD, University of Kansas, Lawrence, KS
- Beatriz M. Fontoura, PhD, University of Texas Southwestern Medical Center, Dallas, TX
- Ana Losada, PhD, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
- Katharine S. Ullman, PhD, Huntsman Cancer Institute, University of Utah, Salt Lake City UT
- Alfred Yergey, PhD, Biomedical Mass Spectrometry Facility, NICHD, Bethesda, MD