ANALYSIS OF THE S PHASE CHECKPOINT IN HIGHER EUKARYOTES

Child Health And Human Development

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Abstract

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. Significantly, both of these pathways have also been directly implicated in the regulation of nuclear trafficking. Our studies of Ran have revealed a surprising role for this protein in mitotic spindle assembly. The nucleotide binding state of Ran is regulated by a GTPase activating protein (RanGAP1) and a guanine nucleotide exchange factor (RCC1). Ran also interacts with a guanine nucleotide dissociation inhibitor (RanBP1) that acts as a cofactor for RanGAP1, increasing the rate of GAP-mediated GTP-Ran hydrolysis. We have found that exogenous RanBP1 protein prolongs mitosis in cycling Xenopus egg extracts, and that spindle assembly is dramatically disrupted under these conditions. Our further investigations have shown that Ran regulates microtubule dynamics in a transport-independent manner that does not require centrioles or chromatin?associated factors. Increased levels of GTP-Ran promote the formation of noncentrosomal microtubule organizing centers (MTOCs), suggesting that some GTP-Ran-dependent step may normally be limiting for MTOC formation in egg extracts. Ran's distinct role in both nuclear transport and spindle assembly may explain the nature of its role as a cell cycle regulator: RanGAP1 is cytosolic during interphase, while RCC1 is chromatin associated, such that nuclear Ran is GTP bound and cytosolic Ran is GDP bound. This asymmetry is important for the directionality in nuclear transport. However, this gradient is broken down during mitosis, and it is possible that the cell cycle machinery may detect dispersion of the Ran gradient as a signal that nuclear envelope breakdown (NEB) has occurred and that spindle assembly should proceed. We are now testing this hypothesis.In other experiments, we have examined the regulation of RanGAP1 and RanBP2 by conjugation with SUMO-1, a small ubiquitin-related modifier. RanBP2 is a large nuclear pore complex (NPC) component that contains multiple RanBP1-related Ran-binding domains. SUMO-1 is conjugated to RanGAP1 and RanBP2 through a pathway that is highly reminiscent of the ubiquitin conjugation pathway. However, SUMO-1 conjugation requires a distinct set of enzymes and may rely upon different regulatory mechanisms. We have cloned the mouse subunits of the heterodimeric E1 enzyme for SUMO-1. We have examined the biochemical properties of these proteins, their subcellular localization and their expression profile during development and the cell cycle. These studies have not only elucidated the properties of the mammalian E1 enzyme for SUMO-1, but in combination with our previous work on the E2 enzyme they will allow us to investigate the regulation and specificity of the SUMO-1 conjugation pathway.