Our goal is to understand how cells send and interpret signals that coordinate events during the cell cycle. Our studies concentrate on two closely linked biochemical pathways, both of which have been genetically implicated in the regulation of mitosis: the Ran GTPase pathway and the SUMO-1 conjugation pathway. Ran-GTP acts as an important indicator for the spatial organization of cells during interphase and mitosis while SUMO-1 regulates Ran through the modification of its GTPase-activating protein, RanGAP1. We are currently examining how these pathways are regulated and how they in turn contribute to the control of mitotic progression.

The Small Ubiquitin-Like Modifier SUMO-1 in Higher Eukaryotes
Anan, Ayaydin, Azuma, Breaux, Cavenagh, Hang, Joseph, Quimby, Tan, Dasso
SUMO-1 is a ubiquitin-like protein that can be conjugated to other proteins in a manner similar to ubiquitin conjugation. Our goal is to understand the function and regulation of SUMO-1 at a molecular level, with a particular focus on the mitotic roles of SUMO-1 and how SUMO-1 controls the Ran GTPase-activating protein, RanGAP1.

SUMO proteases are required for the processing of SUMO-1 before conjugation and for deconjugation of SUMO-1 from other proteins. Two SUMO proteases have been described in budding yeast, 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 have been studying one member of this family in particular: SENP2. We found that full-length human SENP2 associates with nuclear pores in a manner similar to Ulp1 in yeast. The association occurs exclusively with the nuclear face of the pore and requires sequences near the N-terminus of SENP2. We also found that SENP2 binds specifically to Nup153, a nucleoporin localized to the nucleoplasmic face of the nuclear pore, and that the association requires the same domain of SENP2 that mediates its targeting in vivo. Remarkably, a mutant SENP2 protein that is unable to bind to Nup153 is significantly more effective in promoting deconjugation of SUMO-1-conjugated species, indicating that localization of SENP2 to the nuclear pore plays an important role in spatially restricting the activity of this enzyme.

Ran-GTP has an important role in regulating the organization of the cell during both interphase and mitosis (see below). 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 with SUMO-1. SUMO-1 modification causes RanGAP1 to associate with RanBP2, a large nuclear pore protein, and with Ubc9, the E2 enzyme for SUMO-1 conjugation. We have examined the behavior of RanGAP1 during mitosis and found that RanGAP1 associates with mitotic spindles and is particularly concentrated at foci near kinetochores. Association with kinetochores appears soon after nuclear envelope breakdown and persists until late anaphase but is lost coincident with nuclear envelope assembly in telophase. A mutant RanGAP1 protein lacking the capacity to be conjugated to SUMO-1 no longer associated with spindles, indicating that conjugation is essential for mitotic localization of RanGAP1. RanBP2 co-localizes with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. Our findings have shown for the first time that SUMO-1 conjugation is required for mitotic localization of RanGAP1 and indicate that a major role of SUMO-1 conjugation to RanGAP1 may be the spatial regulation of the Ran pathway during mitosis.

Mitotic Roles of Ran GTPase
Arnaoutouv, Dasso
We are interested in the systems required for maintenance of mitotic arrest. To complete division and exit mitosis, cells must degrade the cyclin B protein and thereby inactivate the cdc2 kinase. Cyclin B is targeted for degradation by the anaphase-promoting complex (APC/C) with the help of FZY, an adapter protein whose binding to APC/C is necessary for cyclin ubiquitination. After ubiquitination, cyclin B is rapidly degraded by proteasomes. Cells can be arrested in M phase in response to developmental cues (i.e., Xenopus laevis frog eggs remain arrested in second meiotic metaphase until fertilization; called CSF arrest) or in response to checkpoint signals (i.e., most vertebrate cells arrest in M phase if one or more unattached kinetochores are present; called checkpoint arrest). Both types of arrest can be mimicked in extracts prepared from Xenopus eggs. Addition of calcium eliminates CSF arrest in egg extracts. The mechanism of this release is very similar to the calcium wave triggered by fertilization that promotes destruction of cyclin B and CSF activities and releases the eggs into first interphase. A microtubule-dependent checkpoint can be generated in egg extracts by adding the microtubule destabilizing agent nocodazole and chromosomes. This microtubule-dependent checkpoint is not sensitive to calcium.

To understand the molecular mechanisms that are responsible for generating the two types of arrest, we followed the fate of an I-125–labeled fragment of cyclin B1 in Xenopus egg extracts and observed in vitro reactions with purified components reconstructing ubiquitination by APC/C and FZY, deubiquitination by isopepti-dases, and degradation by proteasomes. We found that exit from mitosis, and therefore degradation of cyclin B, is always accompanied by activation of ubiquitination through APC/C while isopeptidase and proteasomal activities are not changed. After checkpoint activation, APC/C activity was blocked by sequestration of FZY into an inactive complex with checkpoint proteins, thus preventing association of FZY with APC/C. Cyclin B degradation could be restored in checkpoint-arrested extracts with physio-logical levels of baculovirus-produced FZY. While CSF arrest is also mediated by inhibition of APC/C, we found that it cannot be restored by the addition of FZY. To the contrary, our data currently suggest that CSF arrest results from direct inhibition of APC/C by phosphorylation. Taken together, our results indicate that different types of mitotic arrest use different approaches to stabilize Cyclin B.

We are also interested in examining how Ran interacts with mitotic regulatory pathways. Ran is a GTPase that is required for nuclear transport, cell cycle control, mitotic spindle formation, and post-mitotic nuclear assembly. The cytosolic GTPase-activating protein RanGAP1 and the chromatin-bound nucleotide exchange factor RCC1 regulate Ran. The distribution of Ran-GTP provides important spatial information that directs cellular activities throughout the cell cycle. During interphase, the localization of RCC1 and RanGAP1 predicts that nuclear Ran is bound to GTP and cytosolic Ran is bound to GDP. This compartmentalization determines the direction of nuclear transport by promoting the loading and unloading of transport receptors in a manner that is appropriate to the nucleus or cytosol. In mitosis, chromatin-bound RCC1 protein generates a high concentration of Ran-GTP in the vicinity of the chromosomes. In a manner that is essential for the formation of a correct bipolar spindle, Ran-GTP acts to stabilize microtubules (MTs) near the chromosomes. We have observed that the addition of bacterially expressed RCC1, but not an inactive RCC1 mutant, overrides the spindle-assembly checkpoint in a dose-dependent manner. We have further documented that exogenous RCC1 changes the behavior of checkpoint pathway components. Notably, increased concentrations of RCC1 do not release extracts from CSF-mediated arrest. Our results suggest that the mitotic function of Ran GTPase lies not only in formation of the microtubule spindle but also in regulating the checkpoint pathway.