We wish to understand the regulation of cell division in metazoan cells. Our studies focus on two closely linked biochemical pathways that have been implicated in both the regulation of mitosis and nuclear-cytoplasmic trafficking: the SUMO conjugation pathway and the Ran GTPase pathway. SUMO proteins are a conserved family of ubiquitin-like proteins that become covalently conjugated to other cellular proteins. The three SUMO paralogues in mammals all use common enzymes for their conjugation. In recent studies, we investigated the specificity of paralogue utilization, the changing patterns of SUMO conjugation to individual substrates during the cell cycle, and the behavior of enzymes that control SUMO conjugation and deconjugation. We are now extending our studies to understand the enzymology and specificity of particular SUMO conjugation or deconjugation events. The Ran GTPase is required for nucleocytoplasmic trafficking, spindle assembly, nuclear assembly, and cell cycle control. We are investigating the roles of Ran pathway components during mitotic spindle assembly and in the regulation of the metaphase-anaphase transition of higher eukaryotes. Previous studies have demonstrated that Ran plays a critical role in both of these aspects of mitosis, and we are currently attempting to understand this role at the molecular level.
SUMO family small ubiquitin-like modifiers in higher eukaryotes
Ayaydin, Azuma, Kametaka, Mukhopadhyay, Quimby, Wang, Yun, Dasso
Conjugation of SUMO proteins is biochemically similar to ubiquitin conjugation: SUMO proteins are processed to yield a C-terminal di-glycine motif. Processed SUMO proteins form a thioester bond to their activating (E1) enzyme through an ATP-dependent reaction and are transferred to a thioester intermediate with their conjugating (E2) enzyme, Ubc9. Finally, an isopeptide bond is formed between SUMO proteins and their substrates through the cooperative action of Ubc9 and protein ligases (E3). The three broadly expressed human SUMO paralogues have been implicated in a variety of cell functions, including nuclear trafficking, chromosome segregation, chromatin organization, transcription, and RNA metabolism. SUMO-1 is about 45 percent identical to SUMO-2 and SUMO-3, which are 96 percent identical to each other. We are interested in the distinct roles of individual SUMO paralogues within vertebrate cells and the specificity of their conjugation to and deconjugation from their targets.
It is currently unclear whether SUMO-1, -2, and -3 function in ways that are unique, redundant, or antagonistic. Moreover, all three paralogues share common E1 and E2 enzymes while the specificity of SUMO ligases and proteases is not well understood. To address the dynamic properties of SUMO paralogues, we developed stable HeLa-derived cell lines that express biofluorescent SUMO chimeras at levels comparable to those of the endogenous proteins. Through live imaging and photobleaching studies, we have found that SUMO-1 differs from SUMO-2 and SUMO-3 in both its localization and dynamics throughout the cell cycle. In addition, we found significant differences between SUMO-1 dynamics in different subnuclear compartments. Our findings demonstrate that mammalian SUMO paralogues show discrete temporal and spatial patterns of utilization throughout the cell cycle, thus building the case that they are functionally distinct and specifically regulated in vivo.
Given that SUMO proteases can sever the linkage of SUMO proteins to their substrates, it is likely that SUMO modification is highly dynamic in vivo. Both processing and deconjugation are mediated by the same family of SUMO proteases (SENP1, 2, 3, 5, 7 and SUSP1, collectively referred to as SENPs). Relatively little is known, however, about the enzymatic differences and distinct biological roles of these enzymes. Using RNA interference (RNAi), we have selectively suppressed the synthesis of individual SENPs in cell lines stably expressing different GFP-SUMO fusion proteins. We have observed that different GFP-SUMO fusions become re-localized after the depletion of individual SENPs in distinct, paralogue-specific patterns, suggesting that the enzymes show considerable in vivo specificity. Biochemical analysis supports the notion that SENPs show strong paralogue specificity. The findings argue that SENPs are highly specific and that they play a critical role in determining the spectrum of SUMO-conjugated proteins in cells.
In addition to experiments examining the role of SUMO-1 conjugation in the regulation of RanGAP1 (see below), we have sought to identify conjugation targets whose modification is cell cycle–dependent. Our earlier results showed that topoisomerase-II is modified exclusively by SUMO-2/3 during mitosis in Xenopus egg extracts and that SUMO-2/3 conjugation may mobilize topoisomerase-II from mitotic chromatin in a manner important for chromosome segregation at the metaphase-anaphase transition. In recent studies, we found that the SUMO ligase PIASy is specifically required for mitotic conjugation of topoisomerase-II to SUMO-2 in Xenopus egg extracts. PIASy was unique among PIAS family members in its capacity to bind to mitotic chromosomes and recruit the SUMO-conjugating enzyme Ubc9 onto chromatin. The properties are essential as demonstrated by the fact that PIASy mutants that did not bind to chromatin or failed to recruit Ubc9 were functionally inactive. Moreover, PIASy depletion from extracts eliminated essentially all chromosomal accumulation of SUMO-2–conjugated species, suggesting that PIASy is the primary ligase for mitotic chromosomal substrates of SUMO-2. PIASy-dependent SUMO-2–conjugated species concentrated on the inner centromere, and inhibition of PIASy blocked anaphase sister chromatid segregation. Taken together, our observations suggest that PIASy is a critical regulator of mitotic SUMO-2 conjugation for topoisomerase-II and other chromosomal substrates and that its activity may have particular relevance for centromeric functions required for proper chromosome segregation.
Ayaydin F, Dasso M. Distinct in vivo dynamics of vertebrate SUMO paralogues. Mol Biol Cell 2004;15:5208-5218.
Azuma Y, Arnaoutov A, Anan T, Dasso M. PIASy mediates SUMO-2 conjugation of Topoisomerase-II on mitotic chromosomes. EMBO Journal 2005;24:2172-2182.
Azuma Y, Arnaoutov A, Dasso M. SUMO-2 regulates topoisomerase-II in mitosis. J Cell Biol 2003;163:477-487.
Mitotic roles of the Ran GTPase
Arnaoutov, Boyarchuk, Joseph, Mishra, Quimby, Yoon, Dasso; in collaboration with Meier, Yen
The Ran GTPase is required for nuclear assembly, nuclear transport, spindle assembly, and mitotic regulation. The chromatin-associated protein RCC1 is the nucleotide exchange factor for Ran. RanGAP1 is the activating protein for the Ran. Ran-GTP nucleotide hydrolysis also requires a family of Ran-GTP binding proteins that act as RanGAP1 accessory factors. The family includes RanBP1 and RanBP2. Vertebrate RanGAP1 is conjugated to a small ubiquitin-like protein, SUMO-1. Such modification promotes association of RanGAP1 with the interphase nuclear pore complex (NPC) through binding to the nucleoporin RanBP2. During mitosis, RanGAP1 is concentrated at kinetochores in a microtubule- (MT) and SUMO-1–dependent fashion. RanBP2 is also abundantly found on kinetochores in mitosis. Interestingly, plant RanGAP1 is targeted to the NPC during interphase and to the cell plate during mitosis, using mechanisms that are independent of the SUMO pathway. It has been widely hypothesized that the distribution of Ran’s regulators modulate local concentrations of Ran-GTP within cells, spatially directing processes in which Ran has been implicated. We have been examining the mechanisms through which key Ran regulators are localized within mitotic metazoan cells and the functional consequences to cells when such distribution patterns are disrupted.
Using RNA interference to deplete cellular RanBP2, we showed that RanBP2 and RanGAP1 are targeted as a single complex that is both regulated by and essential for stable kinetochore-MT association in mitotic spindles. We also documented that Crm1, a Ran-GTP–binding nuclear export receptor, localizes to kinetochores. Moreover, Crm1 ternary complex assembly is essential for Ran-GTP–dependent kinetochore recruitment of the RanGAP1/RanBP2 complex. Inhibition of Crm1 using the drug Leptomycin B (LMB) causes release of RanGAP1/RanBP2 from kinetochores and the formation of spindles in which continuous MT bundles span the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibers. These findings demonstrate that proper localization of RanGAP1/RanBP2 is essential for definition of kinetochore fibers and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and chromosome segregation in somatic cells.
The spindle assembly checkpoint monitors spindle formation and prevents the onset of the metaphase-anaphase transition until chromosomes are correctly attached and aligned on the metaphase plate. In previous experiments, we documented that the spindle assembly checkpoint can be regulated through Ran-GTP in Xenopus egg extracts. In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the NPCs during interphase, and we examined the relationship of these proteins to the Ran pathway in budding yeast. We found that deletion of yeast MAD1 or MAD2 did not grossly affect steady-state nucleocytoplasmic trafficking or Ran localization. However, yeast with conditional mutations in the yeast Ran GTPase pathway that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. These observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC.
Arnaoutov A, Azuma Y, Ribbeck R, Joseph J, Boyarchuk Y, Dasso M. Crm1 is a mitotic effector of Ran-GTP in somatic cells. Nat Cell Biol 2005;7:626-632.
Arnaoutov A, Dasso M. Ran-GTP regulates kinetochore attachment in somatic cells. Cell Cycle 2005;4:1161-1165.
Jeong SY, Rose A, Joseph J, Dasso M, Meier I. Plant-specific mitotic targeting of RanGAP requires a functional WPP domain. Plant J 2005;42:270-282.
Joseph J, Liu S-T, Jablonski SA, Yen TJ, Dasso M. The RanGAP1/RanBP2 complex is essential for microtubule-kinetochore interactions in vivo. Curr Biol 2004;14:611-617.
Quimby BB, Arnaoutov A, Dasso M. The Ran GTPase regulates Mad2 localization to the nuclear pore complex. Eukaryot Cell 2005;4:274-280.
1Graduate Partnerships Program
Collaborators
Iris Meier, PhD, Ohio State University, Columbus, OH
Tim Yen, PhD, Fox Chase Cancer Center, Philadelphia, PA
For further information, contact mdasso@helix.nih.gov.