Mary Dasso, PhD, Head, Section on Cell Cycle Regulation
Alexei Arnaoutov, PhD, Visiting Fellow
Ai Kametaka, PhD, Visiting Fellow
Ram Kumar Mishra, PhD, Visiting Fellow
Debaditya Mukhopadhyay, PhD, Visiting Fellow
Yonggang Wang, PhD, Visiting Fellow
Hyun-Joo Yoon, PhD, Visiting Fellow
Chawon Yun, PhD, Visiting Fellow
Maia Ouspenskaia, DVM, Biologist/Technician
Yekaterina Boyarchuk, BA, Graduate Student ¹

Our studies focus on two closely linked biochemical pathways that have been implicated in both mitotic regulation and nuclear-cytoplasmic trafficking: the SUMO pathway and the Ran pathway. SUMO proteins are a family of ubiquitin-like proteins that become covalently conjugated to cellular targets. The three mammalian SUMO paralogues 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 controls many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly, and cell cycle progression. We are particularly interested in the role(s) of Ran at mitotic kinetochores, where Ran is essential for both regulation of the spindle assembly checkpoint and assembly of microtubule fibers that attach kinetochores to spindle poles. We are currently focusing on mechanisms that target Ran pathway components to the kinetochore as well as on interactions of Ran pathway components at kinetochores with other proteins that are structural or functional constituents of the interphase nuclear pore.

SUMO family small ubiquitin-like modifiers in higher eukaryotes

Kametaka, Mukhopadhyay, Ouspenskaia, Wang, Yun; in collaboration with Wilkinson

SUMO proteins are a family of ubiquitin-related proteins that become covalently linked to other cellular proteins. While budding yeast has a single SUMO called Smt3p, there are three commonly expressed mammalian SUMO paralogues called SUMO-1, SUMO-2, and SUMO-3. SUMO-2 and SUMO-3 are 96 percent identical while SUMO-1 is roughly 45 percent identical to either SUMO-2 or SUMO-3. Human SUMO paralogues have been implicated in a variety of cell functions, including nuclear trafficking, chromosome segregation, chromatin organization, transcription, and RNA metabolism. The conjugation pathway for SUMO proteins is similar to the ubiquitin conjugation pathway: SUMO proteins are processed by sentrin-specific/ubiquitin-like proteases (SENP/Ulps) to reveal a di-glycine motif at their C-termini. After processing, SUMO proteins undergo ATP-dependent formation of a thioester bond in their activating (E1) enzyme Aos1/Uba2. The activated SUMO proteins are transferred to form a thioester linkage with their conjugating (E2) enzyme Ubc9. Finally, an isopeptide bond is formed between SUMO proteins and substrates through the cooperative action of Ubc9 and protein ligases (E3). The linkage of SUMO proteins to their substrates can be severed by SUMO proteases, strongly suggesting that SUMO modification is highly dynamic in vivo. As with processing, SUMO deconjugation is mediated by SENP/Ulps.

We are interested in the roles of individual vertebrate SUMO paralogues. Using live imaging and photobleaching methods, we demonstrated that mammalian SUMO paralogues show discrete temporal and spatial patterns of utilization, likely indicating that they are functionally distinct and specifically regulated in vivo. One biochemical difference that may be important for unique paralogue behaviors is the paralogues' capacity to form linked chains. As in the case of ubiquitin, Smt3p, SUMO-2, and SUMO-3 can form conjugated chains in vitro and in vivo, although the prevalence and physiological role of SUMO chains have not been established. We have examined the localization, biological function, and enzymatic specificity of SUSP1, which is the largest human SENP/Ulp, and have found that SUSP1 localizes to the nucleoplasm. SUSP1 depletion within cell lines expressing green fluorescent protein EGFP fusions to individual SUMO paralogues caused redistribution of EGFP-SUMO-2 and SUMO-3, particularly into PML bodies. PML bodies are nuclear structures of undefined function that contain the Promyelocytic Leukemia Protein (PML), a major SUMO conjugation substrate. Notably, both the size and number of PML bodies increased after SUSP1 depletion. Further analysis suggested that the change resulted primarily from a deficit of SUMO-2/3 deconjugation activity. We did not observe a comparable redistribution of EGFP-SUMO-1. We investigated the specificity of SUSP1 by using vinyl sulfone (VS) inhibitors and model substrates. We found that SUSP1 has a strong paralogue preference for SUMO-2/3 and that it acts preferentially on substrates containing three or more SUMO-2/3 moieties. Together, our findings argue that, in dismantling highly conjugated SUMO-2 and SUMO-3 species, SUSP1 may play a very specialized role that is critical for PML body maintenance.

Notably, results of other experiments suggest that the PML protein may be not only an important substrate for SUMO conjugation but also a regulator of the pathway. PML was first identified through its fusion to the retinoic acid receptor alpha (RARα) in acute promyelocytic leukemia (APL) patients. In APL cell lines that express PML:RARα, we observed that a subset of proteins showed retinoic acid-dependent changes in their SUMO modification. Interestingly, PML and PML:RARα expression in yeast both stimulated hSUMO-1 modification but differentially complemented yeast SUMO pathway mutants. Together with additional findings, our data indicate that PML and PML:RARα may regulate SUMO conjugation and suggest that fusion of RAR to PML may affect such regulation. It is obviously of interest to speculate that such changes in SUMO modification patterns may contribute to the onset of APL.

Finally, we examined SUMO-2/3-specific modification of topoisomerase-II in mitotic Xenopus egg extracts and found that the SUMO ligase PIASy is specifically required for this conjugation. 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. In combination, our observations suggest that PIASy is a critical regulator of mitotic SUMO-2 conjugation and that its activity may have particular relevance for centromeric functions required for proper chromosome segregation.

Mitotic roles of the Ran GTPase

Arnaoutov, Boyarchuk, Mishra, Yoon; in collaboration with Fontoura, Forbes, Ullman

The Ran GTPase is required for many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly, and cell cycle control. Ran's nucleotide exchange factor is called RCC1, and it remains chromatin-associated throughout the cell cycle. Ran's GTPase-activating protein is called RanGAP1. Ran-GTP nucleotide hydrolysis also requires a family of Ran-GTP-binding proteins, which act as RanGAP1 accessory factors. This family includes the RanBP1 protein and the nucleoporin RanBP2. SUMO-1 conjugation of RanGAP1 promotes its association with the interphase nuclear pore complex (NPC) through binding to RanBP2. RanBP2 and RanGAP1 remain associated in mitosis and target to kinetochores as a single complex in a microtubule-dependent fashion. Our work suggests that Ran plays two important roles at mitotic kinetochores, namely, it is essential for regulation of the spindle assembly checkpoint and for assembly of microtubule fibers that attach kinetochores to spindle poles.

We have shown that the RanBP2/RanGAP1 complex is both regulated by and important for stable kinetochore-MT association in mitotic spindles. We found that Crm1, a Ran-GTP-binding nuclear export receptor, localizes to kinetochores. Inhibition of Crm1 with the drug Leptomycin B 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 critical for the definition of kinetochore fibers and for chromosome segregation at anaphase. Thus, Crm1 is a novel Ran-GTP effector for mitotic spindle assembly and chromosome segregation in somatic cells. We are currently attempting to identify kinetochore components required for the Crm1-mediated recruitment of the RanGAP1/RanBP2 complex and to understand this mechanism at the molecular level.

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. We examined the relationship of these proteins to the Ran pathway in budding yeast and 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 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. Our investigations yielded the first evidence that Ran affects the localization of Mad2p to the NPC.

Targeting of the RanGAP1/RanBP2 complex to mitotic kinetochores and association of Mad1 and Mad2 to the interphase NPC both suggest an intimate relationship between kinetochores and nuclear pores. Our collaborators Douglass Forbes and Beatriz Fontoura have shown that the Nup107-160 complex, which includes nine nucleoporins, localizes at mitotic spindles and kinetochores in mammalian cells. In collaborative studies, they have further shown that the Nup107-160 complex is required for spindle assembly in Xenopus egg extracts. On the other hand, the capacity of egg extracts to polymerize microtubules in response to elevated Ran-GTP levels appears to be intact in the absence of the Nup107-160 complex. These findings do not exclude the possibility that the Nup107-160 complex may be an upstream regulator of Ran-GTP, but they do argue against the notion that the complex works as a Ran effector in spindle formation. Notably, the Nup107-160 complex is not required for either activation of the spindle checkpoint in egg extracts or checkpoint silencing by increased levels of Ran-GTP. We are extending these studies by examining the mitotic roles of other nucleoporin complexes and investigating the complexes' possible function as mitotic Ran effectors.

¹ Graduate Partnerships Program

COLLABORATORS

Beatriz M.A. Fontoura, PhD, University of Texas Southwestern Medical Center, Dallas, TX
Douglass J. Forbes, PhD, University of California, San Diego, La Jolla, CA
Katharine S. Ullman, PhD, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT
Keith D. Wilkinson, PhD, Emory University, Atlanta, GA

For further information, contact mdasso@helix.nih.gov.