SCCR | SETE | SNCGE | SPB | UCT | UCSF | SMM | UNC | Main Page
MITOTIC REGULATION IN HIGHER EUKARYOTES
BY RAN AND SUMO-1
|
Mary Dasso, PhD, Head, Section on Cell Cycle Regulation Tadashi Anan, PhD, Visiting Fellow |
|
|
We wish to understand the cell cycle checkpoints that regulate mitosis. Our studies concentrate on two closely linked biochemical pathways that have been implicated in both the regulation of mitosis and nuclear-cytoplasmic trafficking: the Ran GTPase pathway and the SUMO conjugation pathway. SUMO proteins are a conserved family of ubiquitin-like proteins that become covalently conjugated to cellular proteins in a manner similar to ubiquitin. In mammals, three SUMO isoforms all use common enzymes for their conjugation. In recent studies, we have demonstrated that the spindle checkpoint is responsive to Ran-GTP and that Ran plays a pivotal role in the metaphase-anaphase transition. We are currently investigating how, during mitotic exit, Ran-GTP contributes to activation of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase responsible for targeting key mitotic regulators for proteaosomal proteolysis. We have also shown that topoisomerase-II is a major mitotic substrate of SUMO-2 conjugation during mitosis. Inhibition of SUMO conjugation causes defects in chromatid separation. In conjunction with our earlier experiments showing that SUMO-1 conjugation regulates the Ran pathway in mitosis through RanGAP1, our observations imply that conjugation of SUMO family proteins to critical mitotic substrates may coordinate multiple events during mitotic progression. The small ubiquitin-like modier SUMO-1 in higher eukaryotes Anan, Ayaydin, Azuma, Hang, Joseph, Tan, Dasso Fission and budding yeast each contain a single SUMO family protein, and the SUMO proteins have been implicated in the regulation of the cell cycle in both organisms. In the three SUMO isoforms in mammals, the conjugation pathway for all isoforms is similar to the ubiquitin conjugation pathway: SUMO proteins must be processed to yield a C-terminal di-glycine motif. After processing, the rst step in the SUMO conjugation pathway is the ATP-dependent formation of a thioester bond between SUMO proteins and their activating (E1) enzyme. The second step is the formation of a thioester bond between SUMO proteins and their conjugating (E2) enzyme, Ubc9. In the last step, an isopeptide bond is formed between SUMO proteins and substrates through the cooperative action of Ubc9 and protein ligases (E3). To understand the biological role(s) of this pathway, particularly with respect to mitosis and cell cycle progression, we are examining SUMO conjugation targets and SUMO pathway enzymes in a number of ways. We used Xenopus egg extracts to examine cell cycle-dependent changes in SUMO-conjugated proteins. We found a set of high molecular weight, chromatin-dependent mitotic SUMO-containing species, which protein sequencing revealed to be SUMO-conjugated topoisomerase-II. Topoisomerase-II is modified exclusively by SUMO-2/3 during mitosis under normal circumstances; the modication is maximal in metaphase, followed by rapid deconjugation during anaphase. The differential extraction properties of modified and unmodified topoisomerase-II suggest that SUMO-2/3 conjugation may mobilize topoisomerase-II from mitotic chromatin in a manner that is important for chromosome segregation. The results of analyses using dominant negative forms of Ubc9 and topoisomerase-II inhibitors are further consistent with this idea. Together, our findings indicate that SUMO-2/3 conjugation of topoisomerase-II is important for remodeling of mitotic chromosomes at the metaphase-anaphase transition and that failure of such remodeling could be expected to cause high levels of chromosome mis-segregation in vivo. Ran-GTP has an important role in regulating the organization of the cell during both interphase and mitosis (see "Mitotic roles of Ran GTPase"). Given this role, knowledge of the distribution of Ran regulators will be essential for understanding the control and function of this pathway. In metazoans, RanGAP1 is conjugated to SUMO-1. SUMO-1 modification causes RanGAP1 to associate with Ubc9 and RanBP2, a large nuclear pore protein with Ran-GTP binding domains and a SUMO E3 ligase domain. In previous studies, we examined the behavior of RanGAP1 during mitosis and found that RanGAP1 associates with kinetochores through a SUMO-1-dependent mechanism. RanBP2 co-localized with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. Recently, we have examined the structural requirements for targeting RanGAP1 and RanBP2 as well as their function in mitosis. We found that the RNAi against RanBP2 displaced RanGAP1 from kinetochores, supporting the notion that these proteins target to kinetochores as part of a single complex. Both proteins were displaced after RNAi-mediated depletion of integral kinetochore components, likely indicating that they require intact kinetochore structures to localize appropriately. By contrast, peripheral kinetochore proteins were not essential for the correct targeting of either protein. Cells depleted of RanBP2 show abnormalities in both spindle formation and mitotic progression, substantiating the importance of their function during mitosis. SUMO proteases catalyze the processing of SUMO-1 before the conjugation and deconjugation of SUMO-1 from other proteins. Budding yeast has two SUMO proteases, Ulp1p and Ulp2p/Smt4p. Ulp1p is concentrated near the nuclear periphery and interacts with nuclear pore components, as demonstrated in two-hybrid assays. ULP1 is an essential gene, and temperature-sensitive Ulp1p mutants arrest at the G2/M transition of the cell cycle. In mammals, there are at least seven SUMO protease family members. We are systematically investigating these enzymes and have previously shown that one mammalian SUMO protease (SENP2) associates with the nuclear pore through an interaction between its N-terminus and Nup153. More recently, our analysis of SENP protease suggests that other proteases also associate with the nuclear pore but use different binding sites. Together with earlier findings regarding the association of conjugating enzymes with the nuclear pore (Ubc9 and RanBP2), our results confirm that the nuclear pore is a critical site of SUMO pathway regulation in the cell. Azuma A, Arnaoutov A, Dasso M. SUMO-2 regulates topoisomerase-II in mitosis. J Cell Biol 2003;163:477-487. Azuma Y, Dasso M. A new clue at the nuclear pore: RanBP2 is an E3 enzyme for SUMO-1. Dev Cell 2002;2:130-131. Hang J, Dasso M. Association of the human SUMO-1 protease SENP2 with the nuclear pore. J Biol Chem 2002;277:19961-19966. Joseph J, Tan SH, Karpova TS, McNally JG, Dasso M. SUMO-1 targets RanGAP1 to the mitotic spindle. J Cell Biol 12002;56:595-602. Mitotic roles of Ran GTPase Arnaoutov, Breaux, Kametaka, Quimby, Yun, Dasso Ran is a small GTPase required for nucleocytoplasmic trafficking, spindle assembly, nuclear assembly, and cell cycle control. The nucleotide exchange factor for Ran, RCC1, is a chromatin-associated protein. The GTPase-activating protein for Ran, RanGAP1, is cytoplasmic during interphase. During mitosis, the bulk of RanGAP1 is broadly distributed, although a significant fraction of RanGAP1 becomes associated with kinetochores (see previous section). Ran-GTP nucleotide hydrolysis also requires a family of accessory proteins. The best-characterized member of this family is mammalian RanBP1, which is distributed to the cytosol during interphase. In vitro, RanBP1 increases the rate of RanGAP1-mediated Ran-GTP hydrolysis by about an order of magnitude. RanBP1 also promotes dissociation of Ran-GTP from transport receptors, whose binding would otherwise block RanGAP-mediated GTP hydrolysis. It has been widely hypothesized that the distribution of Ran's regulators modulates local concentrations of Ran-GTP within the cell, spatially directing the many processes in which Ran has been implicated. Ran's primary known effectors are a set of Ran-GTP binding proteins that were originally described as nuclear transport receptors. Ran-GTP binding regulates association between these proteins and their transport cargoes.
Defects in the Ran pathway disrupt both the onset and completion of mitosis, although Ran's function in cell cycle progression had not been clearly distinguished from its roles in nuclear transport and spindle assembly. We were therefore interested in examining more closely Ran's role in mitotic regulation. Mitosis is tightly controlled in eukaryotes by the activity of cyclin B and securin. Both cyclin B and securin are ubiquitinated at the metaphase-anaphase transition by the APC/C E3 ligase, working in association with its activators Cdc20/FZY and Cdh1/FZR. In the presence of misassembled spindles, with kinetochores that are unattached or lack tension from spindle microtubules, the onset of anaphase is delayed through activation of a spindle assembly checkpoint. This checkpoint pathway prevents APC/CFZY activation and thereby stabilizes APC/CFZY substrates. After all the chromosomes have become attached and aligned within the mitotic spindle, the checkpoint is turned off, APC/CFZY becomes active, and anaphase commences. Components of the spindle assembly checkpoint include Mad1, Mad2, Mps1, Bub1, Bub3, BubR1, and CENP-E.
We have examined the role of Ran in regulating mitotic checkpoints using Xenopus egg extracts, a well-established model system for checkpoint control. During normal cell cycles in cycling egg extracts, we find that the amount of chromatin-associated RCC1 increases dramatically at the onset of cyclin B destruction. Moreover, moderate levels of exogenous RCC1 protein abrogate mitotic spindle checkpoint arrest and allow cyclin B destruction in extracts containing nuclei plus nocodazole, a microtubule depolymerizing agent. We find that the spindle assembly checkpoint in Xenopus is characterized by decreased APCFZY activity and that the addition of RCC1 to extracts with the activated checkpoint restores APCFZY activity to control levels. To determine the mechanism through which RCC1 abrogates checkpoint arrest, we examined the localization of mitotic regulators, including Mad2, CENP-E, Bub1, and Bub3. We found that these proteins are mislocalized away from kinetochores in nocodazole-treated extracts after the addition of high levels of RCC1 protein. Interestingly, displacement of Bub1 and Bub3 from kinetochores could be reversed by the addition of recombinant RanGAP1 protein, suggesting that their behavior responds directly to Ran-GTP levels. Taken together, our results indicate that the Ran pathway is normally regulated in a highly dynamic manner during mitosis. The transitions of the Ran pathway can be mimicked by the addition of exogenous RCC1 protein, triggering the metaphase-anaphase transition prematurely in the presence of unattached kinetochores. Our observations suggest that changes in RCC1's chromosomal dynamics may be a critical link in the chain of events between completion of metaphase spindle assembly and mitotic exit.
Arnaoutov A, Dasso M. The Ran GTPase regulates kinetochore function. Dev Cell 2003;5:99-111. Dasso M. The Ran GTPase: theme and variations. Curr Biol 2002;12:R502-508. Joseph J, Dasso M. Cellular roles of the Ran GTPase. In: Hamm H, ed. Handbook of Cellular Signaling. San Diego: Academic Press, 2003; in press. Quimby BB, Dasso M. The small GTPase Ran: interpreting the signs. Curr Opin Cell Biol 2003;15:338-344.
For further information, contact mdasso@helix.nih.gov |
|
